Methods of treating cholangiocarcinoma

ABSTRACT

Methods and compositions for treating cholangiocarcinoma.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/754,509, filed Jan. 18, 2013 and U.S. Provisional Application No.61/756,372, filed Jan. 24, 2013, the contents of which are herebyincorporated by reference in their entirety.

BACKGROUND

Cancer represents the phenotypic end-point of multiple genetic lesionsthat endow cells with a full range of biological properties required fortumorigenesis. Indeed, a hallmark genomic feature of many cancers is thepresence of numerous complex chromosome structural aberrations,including translocations, intra-chromosomal inversions, point mutations,deletions, gene copy number changes, gene expression level changes, andgermline mutations, among others.

Cholangiocarcinoma is a cancer that includes mutated epithelial cellsthat originate in the bile ducts. Cholangiocarcinoma is a relativelyrare neoplasm that is classified as an adenocarcinoma (a cancer thatforms glands or secretes significant amounts of mucins). It has anannual incidence rate of about 1-2 cases per 100,000 in the Westernworld, but rates of cholangiocarcinoma have been rising worldwide overthe past several decades (Landis S. et al. (1998) CA Cancer J Clin 48(1): 6-29; Patel T (2002) BMC Cancer 2: 10. doi:10.1186/1471-2407-2-10).

Cancer of the bile ducts can arise within the liver as an intrahepaticcholangiocarcinoma (ICC) or originate from extrahepatic bile ducts as abile duct carcinoma, also referred to as an extra-hepaticcholangiocarcinoma. ICC is the second most common primary hepaticmalignancy after hepatocellular carcinoma (HCC), and accounts for 3% ofthe malignant tumors of the gastrointestinal system and 15% of primaryhepatic malignancies. Because ICC has a routine histologic appearance ofan adenocarcinoma, the diagnosis of ICC on a liver biopsy requires animmunohistochemical (IHC) study of the tumor and a thorough clinicalworkup including imaging studies to rule out a metastatic adenocarcinomato the liver.

Numerous studies have indicated that the incidence and mortality fromICC are increasing worldwide. ICC is associated with primary sclerosingcholangitis, parasitic biliary infection, polycystic disease of theliver, congenital intrahepatic bile duct dilatation (Caroli's Disease),congenital hepatic fibrosis, and choledochal cysts. Chronic Hepatitis Cinfection is an established cause of ICC with some studies describing amore than 300 fold increase in ICC incidence in patients withlong-standing Hepatitis C infections. ICC has also been associated withcigarette smoking, alcohol consumption and exposure to a variety oftoxins and chemical carcinogens. The onset of symptoms of ICC are oftenvague, typically arise late in the course of the disease and includeabdominal pain, anorexia and palpable abdominal mass lesions. Thus, themedian survival for ICC is less than 6 months for inoperable tumors andonly 20 to 40% for patients who undergo surgery and achieve clearmargins.

Cholangiocarcinoma is considered to be an incurable and rapidly lethalmalignancy, unless both the primary tumor and any metastases can befully resected (removed surgically). No potentially curative treatmentexists at this time except surgery; however, most patients have advancedstage disease at presentation and are inoperable at the time ofdiagnosis. Cholangiocarcinoma has near-100% fatality due to attendantliver complications from the damage to the organ. Patients withcholangiocarcinoma are generally managed with chemotherapy, radiationtherapy, and other palliative care measures.

Thus, the need still exists for identifying novel genetic lesionsassociated with cancers such as cholangiocarcinomas. Such geneticlesions can be an effective approach to develop compositions, methodsand assays for evaluating and treating cancer patients.

SUMMARY

The invention is based, at least in part, on the discovery, incholagiocarcinomas, of novel rearrangement events that give rise toalterations in a fibroblast growth factor receptor 2 (FGFR2) gene or aneurotrophic tyrosine receptor kinase (NTRK I)) gene. In certainembodiments, the alteration is chosen from a translocation, a deletion,an inversion, a rearrangement, or an amplification of, an FGFR2 gene orthe NTRK gene. For example, the alteration can be chosen from analteration described in Table 1 and FIGS. 1A-1C. In one embodiment, thealteration includes a fragment of an FGFR2 gene or the NTRK1 gene, e.g.,as exemplified in Table 1, FIGS. 1A-1C and FIGS. 2-17 . Thus, theinvention provides new insights into the treatment of these cancers,such as cholangiocarcinomas. Therefore, described herein are methods fortreating a cholangiocarcinoma carcinoma, including intrahepaticcholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma, as well asnovel FGFR2 and NTRK1 molecules (e.g., fusion molecules); methods andreagents for identifying, assessing or detecting an alteration in anFGFR2 and/or NTRK1.

Accordingly, in one aspect, the invention features a method of treatinga subject having a cholangiocarcinoma. The method includes administeringto the subject an effective amount of an agent (e.g., a therapeuticagent) that targets, antagonizes or inhibits an FGFR2 or NTRK1 (e.g., anFGFR2 or NTRK1 gene product, e.g., an FGFR2 or NTRK1 protein), therebytreating the subject.

In another aspect, the invention features, a method of treating asubject having a cholangiocarcinoma. The method includes administeringto the subject an effective amount of a kinase inhibitor (e.g., atyrosine kinase inhibitor), thereby treating the subject.

In one embodiment, the method further includes acquiring knowledge ofone or both of:

-   -   (i) the presence (or absence) of an alteration in FGFR2 gene        product, e.g., an FGFR2 protein; or    -   (ii) the presence (or absence) of an alteration in NTRK1 gene        product, e.g., an NTRK1 protein,        -   in the subject, or a cancer or tumor sample from the            subject.

In another embodiment, the method further includes identifying thesubject, or a cancer or tumor sample from the subject, as having one orboth of:

-   -   (i) the presence (or absence) of an alteration in FGFR2 gene        product, e.g., an FGFR2 protein; or    -   (ii) the presence (or absence) of an alteration in NTRK1 gene        product, e.g., an NTRK1 protein.

In certain embodiments, the presence of the FGFR2 or NTRK1 alteration,or both, in the subject is indicative that the subject is likely torespond to the agent.

In yet other embodiments, the agent is administered responsive to adetermination of the presence of the FGFR2 or NTRK1 alteration, or both,in the subject, or the cancer or tumor sample from the subject.

Cholangiocarcinoma

In certain embodiments, the cholangiocarcinima comprises one or moremutated cells that originate in the bile duct. In certain embodiments,the cholangiocarcinoma is chosen from an intrahepatic cholangiocarcinomaor an extrahepatic cholangiocarcinoma. In other embodiments, thecholangiocarcinoma comprises, or is identified as having, an alterationthat is chosen from a translocation, a deletion, an inversion, arearrangement, or an amplification of, an FGFR2 gene or the NTRK gene.In one embodiment, the cholangiocarcinoma comprises, or is identified ashaving, an alteration chosen from an alteration described in Table 1 orFIGS. 1A-1C. In one embodiment, the cholangiocarcinoma comprises, or isidentified as having, an alteration includes a fragment of an FGFR2 geneor the NTRK1 gene, e.g., as exemplified in Table 1, FIGS. 1A-1C andFIGS. 2-17 . In yet other embodiments, the cholangiocarcinoma comprises,or is identified as having, a fusion molecule of FGFR2; e.g., a fusionmolecule chosen from FGFR2-TACC3, FGFR2-KIAA1598, BICC1-FGFR2,FGFR2-BICC1, PARK2-FGFR2, FGFR2-NOL4, or ZDHHC6-FGFR2 as described,e.g., in Table 1, FIGS. 1A-1C and FIGS. 2-17 . In other embodiments, thecholangiocarcinoma comprises, or is identified as having, arearrangement or an amplification of FGFR2 as described, e.g., in Table1, FIGS. 1A-1C and FIGS. 2-17 .

In certain embodiments, the alteration in FGFR2 results in upregulation,increased activity (e.g., increased transformative or oncogenicactivity, kinase activity and/or dimerization), and/or increased levelof an FGFR2 gene product (e.g., an FGFR2 protein), compared to awildtype activity of FGFR2.

Subjects

In certain embodiments, the subject has an alteration in FGFR2 or NTRK1,or both, e.g., the subject has a cholangiocarcinoma comprising analteration in FGFR2 or NTRK1, or both, e.g., as described herein. Inother embodiments, the subject is identified, or has been previouslyidentified, as having a cholangiocarcinoma (e.g., an intrahepaticcholangiocarcinoma (ICC) or an extrahepatic cholangiocarcinoma)comprising an alteration in FGFR2 or NTRK1, or both, e.g., as describedherein. In other embodiments, the subject has, or is identified ashaving, an alteration that is chosen from a translocation, a deletion,an inversion, a rearrangement, or an amplification of, an FGFR2 gene orthe NTRK gene. In one embodiment, the subject has, or is identified ashaving, an alteration chosen from an alteration described in Table 1 orFIGS. 1A-1C. In one embodiment, the subject has, or is identified ashaving, an alteration includes a fragment of an FGFR2 gene or the NTRK1gene, e.g., as exemplified in Table 1, FIGS. 1A-1C and FIGS. 2-17 . Inyet other embodiments, the subject has, or is identified as having, afusion molecule of FGFR2; e.g., a fusion molecule chosen fromFGFR2-TACC3, FGFR2-KIAA1598, BICC1-FGFR2, FGFR2-BICC1, PARK2-FGFR2,FGFR2-NOL4, or ZDHHC6-FGFR2 as described, e.g., in Table 1, FIGS. 1A-1Cand FIGS. 2-17 . In other embodiments, the subject has, or is identifiedas having, a rearrangement or an amplification of FGFR2 as described,e.g., in Table 1, FIGS. 1A-1C and FIGS. 2-17 .

In one embodiment, the subject is a human. In one embodiment, thesubject has, or is at risk of having a cholangiocarcinoma (e.g., acholangiocarcinoma as described herein) at any stage of disease, e.g.,Stage I, II, IIIA-IIIC or IV of intrahepatic cholangiocarcinoma; Stage0, IA-IB, IIA-IIB, III or IV of extrahepatic cholangiocarcinoma; or ametastatic cancer. In other embodiments, the subject is a cancerpatient, e.g., a patient having a cholangiocarcinoma as describedherein.

In one embodiment, the subject is undergoing or has undergone treatmentwith a different (e.g., non-FGFR2 or non-NTRK1) therapeutic agent ortherapeutic modality. In one embodiment, the non-FGFR2 or non-NTRK1therapeutic agent or therapeutic modality is a chemotherapy,immunotherapy, or a surgical procedure. In one embodiment, the non-FGFR2or non-NTRK1 therapeutic agent or therapeutic modality comprises one ormore (or all) of: a surgical procedure, flurouracil (e.g., 5-FU,Adrucil, Efudex), doxorubicin (Adriamycin, Rubex), gemcitabine (e.g.,Gemzar) and/or cisplatin (Platinol).

In one embodiment, responsive to the determination of the presence ofthe FGFR2 or NTRK1 alteration, the different therapeutic agent ortherapeutic modality is discontinued. In yet other embodiments, thesubject has been identified as being likely or unlikely to respond tothe different therapeutic agent or therapeutic modality.

In certain embodiments, the subject has participated previously in aclinical trial, e.g., a clinical trial for a different (e.g., non-FGFR2or non-NTRK1) therapeutic agent or therapeutic modality. In otherembodiments, the subject is a cancer patient who has participated in aclinical trial, e.g., a clinical trial for a different (e.g., non-FGFR2or non-NTRK1) therapeutic agent or therapeutic modality.

Agents

In certain embodiments, the agent (e.g., the therapeutic agent) used inthe methods targets and/or inhibits FGFR2 or NTRK1 (e.g., a FGFR2 orNTRK1 gene or gene product as described herein). In one embodiment, theagent binds and inhibits FGFR2 or NTRK1. In one embodiment, the agent isa reversible or an irreversible FGFR2 inhibitor. In certain embodiments,the agent is a pan-FGFR2 inhibitor.

In one embodiment, the agent is an antibody molecule, e.g., ananti-FGFR2 or NTRK1 antibody molecule (e.g., a monoclonal or abispecific antibody), or a conjugate thereof (e.g., an antibody to FGFR2or NTRK1 conjugated to a cytotoxic agent (e.g., mertansine DM1)).

In one embodiment, the agent is a kinase inhibitor. In one embodiment,the kinase inhibitor is chosen from: a multi-specific kinase inhibitor,an FGFR2 inhibitor (e.g., a pan-FGFR2 inhibitor), an NTRK1 inhibitor,and/or a small molecule inhibitor that is selective for FGFR2 or NTRK1;and/or a FGFR2 or NTRK1 cellular immunotherapy.

In an embodiment, the therapeutic agent is chosen from a kinaseinhibitor; a multi-specific kinase inhibitor; an FGF receptor inhibitor(e.g., a pan FGFR2 inhibitor); an antibody molecule (e.g., a monoclonalantibody) against FGFR2; and/or a small molecule (e.g., kinase)inhibitor that is selective for FGFR2 or NTRK1.

In an embodiment the therapeutic agent is selected from antisensemolecules, ribozymes, RNAi, triple helix molecules that hybridize to anucleic acid encoding the fusion, or a transcription regulatory regionthat blocks or reduces mRNA expression of FGFR2 or NTRK1.

In an embodiment the kinase inhibitor is chosen from: a kinaseinhibitor; a multi-specific kinase inhibitor; an FGF receptor inhibitor(e.g., a pan FGFR2 inhibitor); and/or a kinase inhibitor that isselective for FGFR2 or NTRK1.

In an embodiment, the therapeutic agent is chosen from: Regorafenib;Ponatinib; AZD-2171 (Cediranib); AZD-4547; BGJ398; BIBF1120; Brivanib;Dovitinib; ENMD-2076; JNJ42756493; Masitinib; Lenvatinib; LY2874455;Pazopanib; PD-173955; R406; PD173074; Danusertib; Dovitinib DilacticAcid; TSU-68; Tyrphostin AG 1296; MK-2461; Brivanib Alaninate;Lestaurtinib; PHA-848125; K252a; AZ-23; and/or Oxindole-3.

In an embodiment, the therapeutic agent is chosen from Regorafenib orPonatinib.

Other features and embodiments of the invention include one or more ofthe following.

In an embodiment, the method includes acquiring knowledge of thepresence of an alteration, e.g., fusion, from Table 1, FIGS. 1A-1C andFIGS. 2-17 in said subject.

In an embodiment the therapeutic agent is administered responsive to thedetermination of presence of the alteration, e.g., fusion, in a tumorsample from said subject.

In an embodiment the determination of the presence of the alteration,e.g., fusion, comprises sequencing.

In an embodiment the subject is undergoing or has undergone treatmentwith a different therapeutic agent or therapeutic modality, e.g., anon-FGFR2 or non-NTRK1 therapeutic agent or therapeutic modality.

In an embodiment responsive to a determination of the presence of thealteration, e.g., fusion, the different therapeutic agent or therapeuticmodality is discontinued.

In an embodiment the different therapeutic agent or therapeutic modalityis a chemotherapy or a surgical procedure. In one embodiment, thenon-FGFR2 or non-NTRK1 therapeutic agent or therapeutic modalitycomprises one or more (or all) of: a surgical procedure, flurouracil(e.g., 5-FU, Adrucil, Efudex), doxorubicin (Adriamycin, Rubex),gemcitabine (e.g., Gemzar) and/or cisplatin (Platinol).

In another aspect, the invention features, a method of determining thepresence of an alteration, e.g., a fusion, disclosed herein incholangiocarcinoma sample, comprising:

directly acquiring knowledge that an alteration, e.g., a fusion nucleicacid molecule, of Table 1, FIGS. 1A-1C and FIGS. 2-17 is present in asample from a subject.

In an embodiment the acquiring step comprises sequencing.

In an embodiment the method further comprises administering a kinaseinhibitor to the subject responsive to the determination of the presenceof the alteration, e.g., the fusion, in the sample from the subject.

The invention also provides, methods of: identifying, assessing ordetecting an alteration, e.g., fusion, of an FGFR2 or an NTRK1, e.g.,that arises in a cholangiocarcinoma. Exemplary alteration, e.g.,fusions, include those summarized in Table 1, FIGS. 1A-1C and FIGS. 2-17, including a fusion of FGFR2 (e.g., an FGFR2 fusion molecule (e.g., agene product or fragment thereof)) to a partner from Table 1, FIGS.1A-1C and FIGS. 2-17 , or a fusion of NTRK1 (e.g., an NTRK1 fusionmolecule (e.g., a gene product or fragment thereof)) to a partner ofTable 1. In one embodiment, the FGFR2 or NTRK1 is fused to a secondgene, or a fragment thereof, e.g., as described in Table 1, FIGS. 1A-1Cand FIGS. 2-17 . In other embodiments, the fusion molecule is chosenfrom FGFR2-TACC3, FGFR2-KIAA1598, BICC1-FGFR2, FGFR2-BICC1, PARK2-FGFR2,FGFR2-NOL4, ZDHHC6-FGFR2, or RABGAP1L-NTRK1, e.g., as described, e.g.,in Table 1, FIGS. 1A-1C and FIGS. 2-17 . Included are fusion molecules;isolated fusions nucleic acid molecules, nucleic acid constructs, hostcells containing the nucleic acid molecules; purified fusionpolypeptides and binding agents; detection reagents (e.g., probes,primers, antibodies, kits, capable, e.g., of specific detection of afusion nucleic acid or protein); screening assays for identifyingmolecules that interact with, e.g., inhibit, fusions, e.g., novel kinaseinhibitors or binders of FGFR2 or NTRK1.

Nucleic Acid Molecules

In one aspect, the invention features an isolated nucleic acid molecule,or an isolated preparation of nucleic acid molecules, that includes agenetic alteration disclosed herein. Such nucleic acid molecules orpreparations thereof can include a genetic alteration described hereinor can be used to detect, e.g., sequence, a genetic alteration disclosedherein. In other embodiments, the alteration of FGFR2 or NRTK1 is chosenfrom an alteration set forth in Table 1, FIGS. 1A-1C and FIGS. 2-17 . Inother embodiments, the fusion nucleic acid molecule is chosen fromFGFR2-TACC3, FGFR2-KIAA1598, BICC1-FGFR2, FGFR2-BICC1, PARK2-FGFR2,FGFR2-NOL4, ZDHHC6-FGFR2, or RABGAP1L-NTRK1, e.g., as described, e.g.,in Table 1, FIGS. 1A-1C and FIGS. 2-17 .

Nucleic Acid Detection and Capturing Reagents

The invention also features a nucleic acid molecule, e.g., nucleic acidfragment, suitable as probe, primer, bait or library member thatincludes, flanks, hybridizes to, which are useful for identifying, orare otherwise based on, a fusion described herein. In certainembodiments, the probe, primer or bait molecule is an oligonucleotidethat allows capture, detection or isolation of a fusion nucleic acidmolecule described herein, e.g., a fusion of FGFR2 to a second gene, orfragment thereof, e.g., BICC1, KIAA1598, TACC3, PARK2, NOL4, orZDHHC6-FGFR2; or a fusion of NTRK1 to a second gene, or a fragmentthereof, e.g., RABGAP1L (e.g., as described in Table 1, FIGS. 1A-1C andFIGS. 2-17 ).

The oligonucleotide can comprise a nucleotide sequence substantiallycomplementary to a fragment of a fusion between partners describedherein nucleic acid molecules described herein. The sequence identitybetween the nucleic acid fragment, e.g., the oligonucleotide, and thetarget sequence need not be exact, so long as the sequences aresufficiently complementary to allow the capture, detection or isolationof the target sequence. In one embodiment, the nucleic acid fragment isa probe or primer that includes an oligonucleotide between about 5 and25, e.g., between 10 and 20, or 10 and 15 nucleotides in length. Inother embodiments, the nucleic acid fragment is a bait that includes anoligonucleotide between about 100 to 300 nucleotides, 130 and 230nucleotides, or 150 and 200 nucleotides, in length.

In one embodiment, the nucleic acid fragment can be used to identify orcapture, e.g., by hybridization, a fusion nucleic acid moleculedescribed herein, e.g., a fusion of FGFR2 to a second gene, or fragmentthereof, e.g., BICC1, KIAA1598, TACC3, PARK2, NOL4, or ZDHHC6; or afusion of NTRK1 to a second gene, or a fragment thereof, e.g., RABGAP1L(e.g., as described in Table 1, FIGS. 1A-1C and FIGS. 2-17 ). Forexample, the nucleic acid fragment can be a probe, a primer, or a bait,for use in identifying or capturing, e.g., by hybridization, a fusiondescribed herein. In one embodiment, the nucleic acid fragment can beuseful for identifying or capturing a fusion breakpoint. In oneembodiment, the nucleic acid fragment hybridizes to a nucleotidesequence that includes a breakpoint, e.g., a breakpoint of the fusion.

The probes or primers described herein can be used, for example, forFISH detection or PCR amplification. In one exemplary embodiment wheredetection is based on PCR, amplification of the fusion junction can beperformed using a primer or a primer pair, e.g., for amplifying asequence flanking the fusion junctions described herein, e.g., themutations or the junction of a chromosomal rearrangement describedherein.

In one embodiment, a pair of isolated oligonucleotide primers canamplify a region containing or adjacent to a position in the fusion. Forexample, reverse primers can be designed to hybridize to a nucleotidesequence within genomic or mRNA sequence of one partner, and the forwardprimers can be designed to hybridize to a nucleotide sequence within theother fusion partner.

In other embodiments, the nucleic acid fragment includes a bait thatcomprises a nucleotide sequence that hybridizes to a fusion nucleic acidmolecule described herein, and thereby allows the capture or isolationsaid nucleic acid molecule. In one embodiment, a bait is suitable forsolution phase hybridization. In other embodiments, a bait includes abinding entity, e.g., an affinity tag, that allows capture andseparation, e.g., by binding to a binding entity, of a hybrid formed bya bait and a nucleic acid hybridized to the bait.

In other embodiments, the nucleic acid fragment includes a librarymember comprising a nucleic acid molecule described herein. In oneembodiment, the library member includes a rearrangement that results ina fusion described herein.

The nucleic acid fragment can be detectably labeled with, e.g., aradiolabel, a fluorescent label, a bioluminescent label, achemiluminescent label, an enzyme label, a binding pair label, or caninclude an affinity tag; a tag, or identifier (e.g., an adaptor, barcodeor other sequence identifier).

Fusion Polypeptides

In another aspect, the invention features a fusion polypeptide (e.g., apurified fusion polypeptide), a biologically active or antigenicfragment thereof, as well as reagents (e.g., antibody molecules thatbind to a fusion polypeptide), methods for modulating a fusionpolypeptide activity and detection of a fusion polypeptide.

In certain embodiments, the fusion polypeptide is chosen fromFGFR2-TACC3, FGFR2-KIAA1598, BICC1-FGFR2, FGFR2-BICC1, PARK2-FGFR2,FGFR2-NOL4, ZDHHC6-FGFR2, or RABGAP1L-NTRK1, e.g., as described, e.g.,in Table 1, FIGS. 1A-1C and FIGS. 2-17 .

In one embodiment, the fusion polypeptide has at least one biologicalactivity of one or both of its partners.

In other embodiments, the nucleic acid molecule includes a nucleotidesequence encoding a fusion polypeptide that includes a fragment of aeach partner of a fusion described herein.

In a related aspect, the invention features fusion polypeptide orfragments operatively linked to heterologous polypeptides to form fusionproteins.

In another embodiment, the fusion polypeptide or fragment is a peptide,e.g., an immunogenic peptide or protein, that contains a fusion junctiondescribed herein. Such immunogenic peptides or proteins can be used toraise antibodies specific to the fusion protein. In other embodiments,such immunogenic peptides or proteins can be used for vaccinepreparation. The vaccine preparation can include other components, e.g.,an adjuvant.

In another aspect, the invention features antibody molecules that bindsto a fusion polypeptide or fragment described herein. In embodiments theantibody can distinguish wild type from fusion.

Detection Reagents and Detection of Mutations

In another aspect, the invention features a detection reagent, e.g., apurified or an isolated preparation thereof. Detection reagents candistinguish a nucleic acid, or protein sequence, having an alteration,e.g., a breakpoint, of a rearrangement, e.g., of a fusion nucleic acidmolecule described herein. Exemplary fusions include a fusion of FGFR2to a second gene, or fragment thereof, e.g., BICC1, KIAA1598, TACC3,PARK2, NOL4, or ZDHHC6; or a fusion of NTRK1 to a second gene, or afragment thereof, e.g., RABGAP1L, e.g., as described, e.g., in Table 1,FIGS. 1A-1C and FIGS. 2-17 .

Detection reagents, e.g., nucleic acid-based detection reagents, can beused to identify mutations in a target nucleic acid, e.g., DNA, e.g.,genomic DNA or cDNA, or RNA, e.g., in a sample, e.g., a sample ofnucleic acid derived from a cholangiocarcinoma. Detection reagents,e.g., antibody-based detection reagents, can be used to identifymutations in a target protein, e.g., in a sample, e.g., a sample ofprotein derived from, or produced by, a cholangiocarcinoma cell.

Nucleic Acid-based Detection Reagents

In an embodiment, the detection reagent comprises a nucleic acidmolecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, comprisingsequence which is complementary with a nucleic acid sequence on a targetnucleic acid (the sequence on the target nucleic acid that is bound bythe detection reagent is referred to herein as the “detection reagentbinding site” and the portion of the detection reagent that correspondsto the detection reagent binding site is referred to as the “targetbinding site”). In an embodiment, the detection reagent binding site isdisposed in relationship to the interrogation position such that binding(or in embodiments, lack of binding) of the detection reagent to thedetection reagent binding site allows differentiation of mutant andreference sequences for an alteration described herein (e.g., analteration or a fusion nucleic acid molecule described in Table 1, FIGS.1A-1C and FIGS. 2-17 ), e.g., a fusion of FGFR2 to a second gene, orfragment thereof, e.g., BICC1, KIAA1598, TACC3, PARK2, NOL4, or ZDHHC6;or a fusion of NTRK1 to a second gene, or a fragment thereof, e.g.,RABGAP1L), from a reference sequence. The detection reagent can bemodified, e.g., with a label or other moiety, e.g., a moiety that allowscapture.

In an embodiment, the detection reagent comprises a nucleic acidmolecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, which, e.g., inits target binding site, includes the interrogation position and whichcan distinguish (e.g., by affinity of binding of the detection reagentto a target nucleic acid or the ability for a reaction, e.g., a ligationor extension reaction with the detection reagent) between a mutation,e.g., a translocation described herein, and a reference sequence. Inembodiments, the interrogation position can correspond to a terminal,e.g., to a 3′ or 5′ terminal nucleotide, a nucleotide immediatelyadjacent to a 3′ or 5′ terminal nucleotide, or to another internalnucleotide, of the detection reagent or target binding site.

In embodiments, the difference in the affinity of the detection reagentfor a target nucleic acid comprising the mutant and that for a targetnucleic acid comprising the reference sequence allows determination ofthe presence or absence of the mutation (or reference) sequence.Typically, such detection reagents, under assay conditions, will exhibitsubstantially higher levels of binding only to the mutant or only to thereference sequence, e.g., will exhibit substantial levels of bindingonly to the mutant or only to the reference sequence.

In embodiments, binding allows (or inhibits) a subsequent reaction,e.g., a subsequent reaction involving the detection reagent or thetarget nucleic acid. E.g., binding can allow ligation, or the additionof one or more nucleotides to a nucleic acid, e.g., the detectionreagent, e.g., by DNA polymerase, which can be detected and used todistinguish mutant from reference. In embodiments, the interrogationposition is located at the terminus, or sufficiently close to theterminus, of the detection reagent or its target binding site, such thathybridization, or a chemical reaction, e.g., the addition of one or morenucleotides to the detection reagent, e.g., by DNA polymerase, onlyoccurs, or occurs at a substantially higher rate, when there is aperfect match between the detection reagent and the target nucleic acidat the interrogation position or at a nucleotide position within 1, 2,or 3 nucleotides of the interrogation position.

In an embodiment, the detection reagent comprises a nucleic acid, e.g.,a DNA, RNA or mixed DNA/RNA molecule wherein the molecule, or its targetbinding site, is adjacent (or flanks), e.g., directly adjacent, to theinterrogation position, and which can distinguish between a mutation,e.g., a translocation described herein, and a reference sequence, in atarget nucleic acid.

In embodiments, the detection reagent binding site is adjacent to theinterrogation position, e.g., the 5′ or 3′terminal nucleotide of thedetection reagent, or its target binding site, is adjacent, e.g.,between 0 (directly adjacent) and 1,000, 500, 400, 200, 100, 50, 10, 5,4, 3, 2, or 1 nucleotides from the interrogation position. Inembodiments, the outcome of a reaction will vary with the identity ofthe nucleotide at the interrogation position allowing one to distinguishbetween mutant and reference sequences. E.g., in the presence of a firstnucleotide at the interrogation position a first reaction will befavored over a second reaction. E.g., in a ligation or primer extensionreaction, the product will differ, e.g., in charge, sequence, size, orsusceptibility to a further reaction (e.g., restriction cleavage)depending on the identity of the nucleotide at the interrogationposition. In embodiments the detection reagent comprises pairedmolecules (e.g., forward and reverse primers), allowing foramplification, e.g., by PCR amplification, of a duplex containing theinterrogation position. In such embodiments, the presence of themutation can be determined by a difference in the property of theamplification product, e.g., size, sequence, charge, or susceptibilityto a reaction, resulting from a sequence comprising the interrogationposition and a corresponding sequence having a reference nucleotide atthe interrogation positions. In embodiments, the presence or absence ofa characteristic amplification product is indicative of the identity ofthe nucleotide at the interrogation site and thus allows detection ofthe mutation.

In embodiments, the detection reagent, or its target binding site, isdirectly adjacent to the interrogation position, e.g., the 5′ or3′terminal nucleotide of the detection reagent is directly adjacent tothe interrogation position. In embodiments, the identity of thenucleotide at the interrogation position will determine the nature of areaction, e.g., a reaction involving the detection reagent, e.g., themodification of one end of the detection reagent. E.g., in the presenceof a first nucleotide at the interrogation position a first reactionwill be favored over a second reaction. By way of example, the presenceof a first nucleotide at the interrogation position, e.g., a nucleotideassociated with a mutation, can promote a first reaction, e.g., theaddition of a complementary nucleotide to the detection reagent. By wayof example, the presence of an A at the interrogation position willcause the incorporation of a T, having, e.g., a first colorimetriclabel, while the presence of a G and the interrogation position willcause the incorporation for a C, having, e.g., a second colorimetriclabel. In an embodiment, the presence of a first nucleotide at thenucleotide will result in ligation of the detection reagent to a secondnucleic acid. E.g., a third nucleic acid can be hybridized to the targetnucleic acid sufficiently close to the interrogation site that if thethird nucleic acid has an exact match at the interrogation site it willbe ligated to the detection reagent. Detection of the ligation product,or its absence, is indicative of the identity of the nucleotide at theinterrogation site and thus allows detection of the mutation.

A variety of readouts can be employed. E.g., binding of the detectionreagent to the mutant or reference sequence can be followed by a moiety,e.g., a label, associated with the detection reagent, e.g., aradioactive or enzymatic label. In embodiments the label comprises aquenching agent and a signaling agent and hybridization results inaltering the distance between those two elements, e.g., increasing thedistance and un-quenching the signaling agent. In embodiments, thedetection reagent can include a moiety that allows separation from othercomponents of a reaction mixture. In embodiments, binding allowscleavage of the bound detection reagent, e.g., by an enzyme, e.g., bythe nuclease activity of the DNA polymerase or by a restriction enzyme.The cleavage can be detected by the appearance or disappearance of anucleic acid or by the separation of a quenching agent and a signalingagent associated with the detection reagent. In embodiments, bindingprotects, or renders the target susceptible, to further chemicalreaction, e.g., labeling or degradation, e.g., by restriction enzymes.In embodiments binding with the detection reagent allows captureseparation or physical manipulation of the target nucleic acid tothereby allow for identification. In embodiments binding can result in adetectable localization of the detection reagent or target, e.g.,binding could capture the target nucleic acid or displace a thirdnucleic acid. Binding can allow for determination of the presence ofmutant or reference sequences with FISH, particularly in the case ofrearrangements. Binding can allow for the extension or other size changein a component, e.g., the detection reagent, allowing distinctionbetween mutant and reference sequences. Binding can allow for theproduction, e.g., by PCR, of an amplicon that distinguishes mutant fromreference sequence.

In an embodiment the detection reagent, or the target binding site, isbetween 5 and 500, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and100, 5 and 50, 5 and 25, 5 and 20, 5 and 15, or 5 and 10 nucleotides inlength. In an embodiment the detection reagent, or the target bindingsite, is between 10 and 500, 10 and 300, 10 and 250, 10 and 200, 10 and150, 10 and 100, 10 and 50, 10 and 25, 10 and 20, or 10 and 15,nucleotides in length. In an embodiment the detection reagent, or thetarget binding site, is between 20 and 500, 20 and 300, 20 and 250, 20and 200, 20 and 150, 20 and 100, 20 and 50, or 20 and 25 nucleotides inlength. In an embodiment the detection reagent, or the target bindingsite, is sufficiently long to distinguish between mutant and referencesequences and is less than 100, 200, 300, 400, or 500 nucleotides inlength.

Preparations of Mutant Nucleic Acid and Uses Thereof

In another aspect, the invention features purified or isolatedpreparations of a neoplastic or tumor cell nucleic acid, e.g., DNA,e.g., genomic DNA or cDNA, or RNA, containing an interrogation positiondescribed herein, useful for determining if a mutation disclosed hereinis present. The nucleic acid includes the interrogation position, andtypically additional fusion sequence on one or both sides of theinterrogation position. In addition the nucleic acid can containheterologous sequences, e.g., adaptor or priming sequences, typicallyattached to one or both terminus of the nucleic acid. The nucleic acidalso includes a label or other moiety, e.g., a moiety that allowsseparation or localization.

In embodiments, the nucleic acid is between 20 and 1,000, 30 and 900, 40and 800, 50 and 700, 60 and 600, 70 and 500, 80 and 400, 90 and 300, or100 and 200 nucleotides in length (with or without heterologoussequences). In one embodiment, the nucleic acid is between 40 and 1,000,50 and 900, 60 and 800, 70 and 700, 80 and 600, 90 and 500, 100 and 400,110 and 300, or 120 and 200 nucleotides in length (with or withoutheterologous sequences). In another embodiment, the nucleic acid isbetween 50 and 1,000, 50 and 900, 50 and 800, 50 and 700, 50 and 600, 50and 500, 50 and 400, 50 and 300, or 50 and 200 nucleotides in length(with or without heterologous sequences). In embodiments, the nucleicacid is of sufficient length to allow sequencing (e.g., by chemicalsequencing or by determining a difference in T_(m) between mutant andreference preparations) but is optionally less than 100, 200, 300, 400,or 500 nucleotides in length (with or without heterologous sequences).Such preparations can be used to sequence nucleic acid from a sample,e.g., a neoplastic or tumor sample. In an embodiment the purifiedpreparation is provided by in situ amplification of a nucleic acidprovided on a substrate. In embodiments the purified preparation isspatially distinct from other nucleic acids, e.g., other amplifiednucleic acids, on a substrate.

In an embodiment, the purified or isolated preparation of nucleic acidis derived from a cholangiocarcinoma.

Such preparations can be used to determine if a sample comprises mutantsequence, e.g., a translocation as described herein. In one embodiment,the translocation includes a breakpoint, e.g., a breakpoint in fusionnucleic acid molecule described herein, e.g., a fusion of FGFR2 to asecond gene, or fragment thereof, e.g., BICC1, KIAA1598, TACC3, PARK2,NOL4, or ZDHHC6; or a fusion of NTRK1 to a second gene, or a fragmentthereof, e.g., RABGAP1L (e.g., an alteration or a fusion nucleic acidmolecule described in Table 1, FIGS. 1A-1C and FIGS. 2-17 ).

In another aspect, the invention features, a method of determining thesequence of an interrogation position for a mutation described herein,comprising:

providing a purified or isolated preparations of nucleic acid or fusionnucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containingan interrogation position described herein, sequencing, by a method thatbreaks or forms a chemical bond, e.g., a covalent or non-covalentchemical bond, e.g., in a detection reagent or a target sequence, thenucleic acid so as to determine the identity of the nucleotide at aninterrogation position. The method allows determining if a mutationdescribed herein is present.

In an embodiment, sequencing comprises contacting the fusion nucleicacid with a detection reagent described herein.

In an embodiment, sequencing comprises determining a physical property,e.g., stability of a duplex form of the fusion nucleic acid, e.g., ™,that can distinguish mutant from reference sequence.

In an embodiment, the fusion nucleic acid is derived from acholangiocarcinoma.

Reaction Mixtures and Devices

In another aspect, the invention features, purified or isolatedpreparations of a fusion nucleic acid, e.g., DNA, e.g., genomic DNA orcDNA, or RNA, containing an interrogation position described herein,useful for determining if a mutation disclosed herein is present,disposed in sequencing device, or a sample holder for use in such adevice. In an embodiment, the fusion nucleic acid is derived from acholangiocarcinoma.

In another aspect, the invention features, purified or isolatedpreparations of a fusion nucleic acid, e.g., DNA, e.g., genomic DNA orcDNA, or RNA, containing an interrogation position described herein,useful for determining if a mutation disclosed herein is present,disposed in a device for determining a physical or chemical property,e.g., stability of a duplex, e.g., ™ or a sample holder for use in sucha device. In an embodiment, the device is a calorimeter. In anembodiment the fusion nucleic acid is derived from a cholangiocarcinoma.

The detection reagents described herein can be used to determine if amutation described herein is present in a sample. In embodiments, thesample comprises a nucleic acid that is derived from acholangiocarcinoma. The cell can be from a neoplastic or a tumor sample,e.g., a biopsy taken from the neoplasm or the tumor; from circulatingtumor cells, e.g., from peripheral blood; or from a blood or plasmasample. In an embodiment, the fusion nucleic acid is derived from acholangiocarcinoma.

Accordingly, in one aspect, the invention features a method of making areaction mixture, comprising:

-   -   combining a detection reagent, or purified or isolated        preparation thereof, described herein with a target nucleic acid        derived from a cholangiocarcinoma which comprises a sequence        having an interrogation position for a mutation described        herein.

In another aspect, the invention features a reaction mixture,comprising:

-   -   a detection reagent, or purified or isolated preparation        thereof, described herein; and    -   a target nucleic acid derived from a cholangiocarcinoma cell        which comprises a sequence having an interrogation position for        a mutation described herein.

In an embodiment of the reaction mixture, or the method of making thereaction mixture: the detection reagent comprises a nucleic acid, e.g.,a DNA, RNA or mixed DNA/RNA, molecule which is complementary with anucleic acid sequence on a target nucleic acid (the detection reagentbinding site) wherein the detection reagent binding site is disposed inrelationship to the interrogation position such that binding of thedetection reagent to the detection reagent binding site allowsdifferentiation of mutant and reference sequences for a mutant describedherein.

In an embodiment of the reaction mixture, or the method of making thereaction mixture, the cholangiocarcinoma is as described herein.

In an embodiment of the reaction mixture, or the method of making thereaction mixture: the alteration, e.g., the mutation is an alteration,e.g., a mutation, described herein, including: a translocation, adeletion, an invention, a rearrangement, an amplification as describedherein (e.g., an alteration as described in Table 1, FIGS. 1A-1C andFIGS. 2-17 ). In one embodiment, the alteration, e.g., mutation, is afusion described herein, e.g., a fusion of FGFR2 to a second gene, orfragment thereof, e.g., BICC1, KIAA1598, TACC3, PARK2, NOL4 or ZDHHC6;or a fusion of NTRK1 to a second gene, or a fragment thereof, e.g.,RABGAP1L).

An alteration, e.g., a mutation described herein, can be distinguishedfrom a reference, e.g., a non-mutant or wildtype sequence, by reactionwith an enzyme that reacts differentially with the mutation and thereference. E.g., they can be distinguished by cleavage with arestriction enzyme that has differing activity for the mutant andreference sequences. E.g., the invention includes a method of contactinga nucleic acid comprising an alteration, e.g., a mutation, describedherein with such an enzyme and determining if a product of that cleavagewhich can distinguish mutant form reference sequence is present.

In one aspect the inventions provides, a purified preparation of arestriction enzyme cleavage product which can distinguish between mutantand reference sequence, wherein one end of the cleavage product isdefined by an enzyme that cleaves differentially between mutant andreference sequence. In an embodiment, the cleavage product includes theinterrogation position.

Protein-based Detection Reagents, Methods, Reaction Mixtures and Devices

A mutant protein described herein can be distinguished from a reference,e.g., a non-mutant or wild-type protein, by reaction with a reagent,e.g., a substrate, e.g, a substrate for catalytic activity or functionalactivity, or an antibody, that reacts differentially with the mutant andreference protein. In one aspect, the invention includes a method ofcontacting a sample comprising a mutant protein described herein withsuch reagent and determining if the mutant protein is present in thesample.

In another embodiment, the invention features, an antibody that candistinguish a mutant protein described herein, e.g., a mutant proteincorresponding to fusion described herein, e.g., a fusion of FGFR2 to asecond gene, or fragment thereof, e.g., BICC1, KIAA1598, TACC3, PARK2,NOL4 or ZDHHC6, or a fusion of NTRK1 to a second gene, or a fragmentthereof, e.g., RABGAP1L, or an associated mutation from a reference,e.g., a non-mutant or wildtype protein (e.g., a fusion polypeptidedescribed in Table 1, FIGS. 1A-1C and FIGS. 2-17 ).

Accordingly, in one aspect, the invention features a method of making areaction mixture comprising combining a detection reagent, or purifiedor isolated preparation thereof, e.g., a substrate, e.g., a substratefor phosphorylation or other activity, or an antibody, described hereinwith a target fusion protein derived from a cholangiocarcinoma cellwhich comprises a sequence having an interrogation position for amutation described herein.

In another aspect, the invention features a reaction mixture,comprising:

-   -   a detection reagent, or purified or isolated preparation        thereof, e.g., a substrate, e.g., a substrate for        phosphorylation or other activity, or an antibody, described        herein; and a target fusion protein derived from a        cholangiocarcinoma cell which comprises a sequence having an        interrogation position for a mutation described herein.

In an embodiment of the reaction mixture, or the method of making thereaction mixture the detection reagent comprises an antibody specificfor a mutant fusion protein described herein.

In an embodiment of the reaction mixture, or the method of making thereaction mixture the cholangiocarcinoma cell.

In an embodiment of the reaction mixture, or the method of making thereaction mixture the mutation is a mutation described herein, including:a translocation event, e.g., a translocation as described herein. In oneembodiment, the mutation is a breakpoint, found in a fusion describedherein, e.g., a fusion of FGFR2 to a second gene, or fragment thereof,e.g., BICC1, KIAA1598, TACC3, PARK2, NOL4, or ZDHHC6; or a fusion ofNTRK1 to a second gene, or a fragment thereof, e.g., RABGAP1L) (e.g., afusion described in Table 1, FIGS. 1A-1C and FIGS. 2-17 ).

Kits

In another aspect, the invention features a kit comprising a detectionreagent as described herein.

Screening Methods

In another aspect, the invention features a method, or assay, forscreening for agents that modulate, e.g., inhibit, the expression oractivity of fusion as described herein. The method includes contacting afusion, or a cell expressing a fusion, with a candidate agent; anddetecting a change in a parameter associated with a fusion, e.g., achange in the expression or an activity of the fusion. The method can,optionally, include comparing the treated parameter to a referencevalue, e.g., a control sample (e.g., comparing a parameter obtained froma sample with the candidate agent to a parameter obtained from a samplewithout the candidate agent). In one embodiment, if a decrease inexpression or activity of the fusion is detected, the candidate agent isidentified as an inhibitor. In another embodiment, if an increase inexpression or activity of the fusion is detected, the candidate agent isidentified as an activator. In certain embodiments, the fusion is anucleic acid molecule or a polypeptide as described herein.

In one embodiment, the contacting step is effected in a cell-freesystem, e.g., a cell lysate or in a reconstituted system. In otherembodiments, the contacting step is effected in a cell in culture, e.g.,a cell expressing fusion (e.g., a mammalian cell, a tumor cell or cellline, a recombinant cell). In yet other embodiments, the contacting stepis effected in a cell in vivo (a -expressing cell present in a subject,e.g., an animal subject (e.g., an in vivo animal model).

In certain embodiments, a method for screening for an agent thatmodulates, e.g., inhibits, the expression or activity of an FGFR2 orNTRK1 alteration, e.g., a fusion, from Table 1, FIGS. 1A-1C and FIGS.2-17 is disclosed. The method includes:

-   -   optionally, determining if the alteration, e.g., fusion, is        present;    -   contacting the alteration, e.g., fusion, (or a host cell        expressing the alteration, e.g., fusion) with a candidate agent;        and    -   detecting a change in a parameter associated with the        alteration, e.g., fusion.

In an embodiment, the parameter is the expression or an activity of theFGFR2 or NTRK1 alteration, e.g., a fusion.

In other embodiments, the parameter is selected from one or more of:

-   -   (i) direct binding of the candidate agent to the FGFR2 or NTRK1        alteration, e.g., a fusion molecule (e.g., fusion polypeptide);    -   (ii) a change in kinase activity;    -   (iii) a change in an activity of a cell containing the        alteration (e.g., the fusion), e.g., a change in proliferation,        morphology or tumorigenicity of the cell;    -   (iv) a change in tumor present in an animal subject, e.g., size,        appearance, proliferation, of the tumor; or    -   (v) a change in the level, e.g., expression level, of the        alteration, e.g., fusion polypeptide or nucleic acid molecule.

Exemplary parameters evaluated include one or more of:

-   -   (i) a change in binding activity, e.g., direct binding of the        candidate agent to a fusion polypeptide; a binding competition        between a known ligand and the candidate agent to a fusion        polypeptide;    -   (ii) a change in kinase activity, e.g., phosphorylation levels        of a fusion polypeptide (e.g., an increased or decreased        autophosphorylation); or a change in a target of an fusion, In        certain embodiments, a change in kinase activity, e.g.,        phosphorylation, is detected by any of Western blot (e.g., using        an anti-fusion antibody, mass spectrometry, immunoprecipitation,        immunohistochemistry, immunomagnetic beads, among others;    -   (iii) a change in an activity of a cell containing a fusion        (e.g., a tumor cell or a recombinant cell), e.g., a change in        proliferation, morphology or tumorigenicity of the cell;    -   (iv) a change in tumor present in an animal subject, e.g., size,        appearance, proliferation, of the tumor; or    -   (v) a change in the level, e.g., expression level, of a fusion        polypeptide or nucleic acid molecule.

In one embodiment, a change in a cell free assay in the presence of acandidate agent is evaluated. For example, an activity of a fusion, orinteraction of a fusion with a downstream ligand can be detected. In oneembodiment, a fusion polypeptide is contacted with a ligand, e.g., insolution, and a candidate agent is monitored for an ability to modulate,e.g., inhibit, an interaction, e.g., binding, between the fusionpolypeptide and the ligand.

In other embodiments, a change in an activity of a cell is detected in acell in culture, e.g., a cell expressing a fusion (e.g., a mammaliancell, a tumor cell or cell line, a recombinant cell). In one embodiment,the cell is a recombinant cell that is modified to express a fusionnucleic acid, e.g., is a recombinant cell transfected with a fusionnucleic acid. The transfected cell can show a change in response to theexpressed fusion, e.g., increased proliferation, changes in morphology,increased tumorigenicity, and/or acquired a transformed phenotype. Achange in any of the activities of the cell, e.g., the recombinant cell,in the presence of the candidate agent can be detected. For example, adecrease in one or more of: proliferation, tumorigenicity, transformedmorphology, in the presence of the candidate agent can be indicative ofan inhibitor of a fusion. In other embodiments, a change in bindingactivity or phosphorylation as described herein is detected.

In yet other embodiment, a change in a tumor present in an animalsubject (e.g., an in vivo animal model) is detected. In one embodiment,the animal model is a tumor containing animal or a xenograft comprisingcells expressing a fusion (e.g., tumorigenic cells expressing a fusion).The candidate agent can be administered to the animal subject and achange in the tumor is detected. In one embodiment, the change in thetumor includes one or more of a tumor growth, tumor size, tumor burden,survival, is evaluated. A decrease in one or more of tumor growth, tumorsize, tumor burden, or an increased survival is indicative that thecandidate agent is an inhibitor.

In other embodiments, a change in expression of a fusion can bemonitored by detecting the nucleic acid or protein levels, e.g., usingthe methods described herein.

In certain embodiments, the screening methods described herein can berepeated and/or combined. In one embodiment, a candidate agent that isevaluated in a cell-free or cell-based described herein can be furthertested in an animal subject.

In one embodiment, the candidate agent is a small molecule compound,e.g., a kinase inhibitor, a nucleic acid (e.g., antisense, siRNA,aptamer, ribozymes, microRNA), an antibody molecule (e.g., a fullantibody or antigen binding fragment thereof that binds to the fusion).The candidate agent can be obtained from a library (e.g., a commerciallibrary of kinase inhibitors) or rationally designed.

Methods for Detecting Fusions

In another aspect, the invention features a method of determining thepresence of a fusion as described herein. In one embodiment, the fusionis detected in a nucleic acid molecule or a polypeptide. The methodincludes detecting whether a fusion nucleic acid molecule or polypeptideis present in a cell (e.g., a circulating cell), a tissue (e.g., atumor), or a sample, e.g., a tumor sample, from a subject. In oneembodiment, the sample is a nucleic acid sample. In one embodiment, thenucleic acid sample comprises DNA, e.g., genomic DNA or cDNA, or RNA,e.g., mRNA. In other embodiments, the sample is a protein sample.

In one embodiment, the sample is, or has been, classified asnon-malignant using other diagnostic techniques, e.g.,immunohistochemistry.

In one embodiment, the sample is acquired from a subject (e.g., asubject having or at risk of having a cancer, e.g., a patient), oralternatively, the method further includes acquiring a sample from thesubject. The sample can be chosen from one or more of: tissue, e.g.,cancerous tissue (e.g., a tissue biopsy), whole blood, serum, plasma,buccal scrape, sputum, saliva, cerebrospinal fluid, urine, stool,circulating tumor cells, circulating nucleic acids, or bone marrow. Incertain embodiments, the sample is a tissue (e.g., a tumor biopsy), acirculating tumor cell or nucleic acid.

In embodiments, the tumor is from a cancer described herein, e.g., ischosen from a cholangiocarcinoma, e.g., an intrahepatic or anextrahepatic cholangiocarcinoma.

In one embodiment, the subject is at risk of having, or has acholangiocarcinoma.

In other embodiments, the fusion is detected in a nucleic acid moleculeby a method chosen from one or more of: nucleic acid hybridizationassay, amplification-based assays (e.g., polymerase chain reaction(PCR)), PCR-RFLP assay, real-time PCR, sequencing, screening analysis(including metaphase cytogenetic analysis by standard karyotype methods,FISH (e.g., break away FISH), spectral karyotyping or MFISH, comparativegenomic hybridization), in situ hybridization, SSP, HPLC ormass-spectrometric genotyping.

In one embodiment, the method includes: contacting a nucleic acidsample, e.g., a genomic DNA sample (e.g., a chromosomal sample or afractionated, enriched or otherwise pre-treated sample) or a geneproduct (mRNA, cDNA), obtained from the subject, with a nucleic acidfragment (e.g., a probe or primer as described herein (e.g., anexon-specific probe or primer) under conditions suitable forhybridization, and determining the presence or absence of the fusionnucleic acid molecule. The method can, optionally, include enriching asample for the gene or gene product.

In a related aspect, a method for determining the presence of a fusionnucleic acid molecule is provided. The method includes: acquiring asequence for a position in a nucleic acid molecule, e.g., by sequencingat least one nucleotide of the nucleic acid molecule (e.g., sequencingat least one nucleotide in the nucleic acid molecule that comprises thefusion), thereby determining that the fusion is present in the nucleicacid molecule. Optionally, the sequence acquired is compared to areference sequence, or a wild type reference sequence. In oneembodiment, the nucleic acid molecule is from a cell (e.g., acirculating cell), a tissue (e.g., a cholangiocarcinoma), or any samplefrom a subject (e.g., blood or plasma sample). In other embodiments, thenucleic acid molecule from a tumor sample (e.g., a tumor or cancersample) is sequenced. In one embodiment, the sequence is determined by anext generation sequencing method. The method further can furtherinclude acquiring, e.g., directly or indirectly acquiring, a sample,e.g., a cholangiocarcinoma.

In another aspect, the invention features a method of analyzing a tumoror a circulating tumor cell. The method includes acquiring a nucleicacid sample from the tumor or the circulating cell; and sequencing,e.g., by a next generation sequencing method, a nucleic acid molecule,e.g., a nucleic acid molecule that includes a fusion as describedherein.

In yet other embodiment, a fusion polypeptide is detected. The methodincludes: contacting a protein sample with a reagent which specificallybinds to a fusion polypeptide; and detecting the formation of a complexof the fusion polypeptide and the reagent. In one embodiment, thereagent is labeled with a detectable group to facilitate detection ofthe bound and unbound reagent. In one embodiment, the reagent is anantibody molecule, e.g., is selected from the group consisting of anantibody, and antibody derivative, and an antibody fragment.

In yet another embodiment, the level (e.g., expression level) oractivity the fusion is evaluated. For example, the level (e.g.,expression level) or activity of the fusion (e.g., mRNA or polypeptide)is detected and (optionally) compared to a pre-determined value, e.g., areference value (e.g., a control sample).

In yet another embodiment, the fusion is detected prior to initiating,during, or after, a treatment in a subject having a fusion.

In one embodiment, the fusion is detected at the time of diagnosis witha cancer. In other embodiment, the fusion is detected at apre-determined interval, e.g., a first point in time and at least at asubsequent point in time.

In certain embodiments, responsive to a determination of the presence ofthe fusion, the method further includes one or more of:

-   -   (1) stratifying a patient population (e.g., assigning a subject,        e.g., a patient, to a group or class);    -   (2) identifying or selecting the subject as likely or unlikely        to respond to a treatment, e.g., a kinase inhibitor treatment as        described herein;    -   (3) selecting a treatment option, e.g., administering or not        administering a preselected therapeutic agent, e.g., a kinase        inhibitor as described herein; or    -   (4) prognosticating the time course of the disease in the        subject (e.g., evaluating the likelihood of increased or        decreased patient survival).

In certain embodiments, responsive to the determination of the presenceof a fusion, the subject is classified as a candidate to receivetreatment with a therapy disclosed herein, e.g., from Table 2. In oneembodiment, responsive to the determination of the presence of a fusion,the subject, e.g., a patient, can further be assigned to a particularclass if a fusion is identified in a sample of the patient. For example,a patient identified as having a fusion can be classified as a candidateto receive treatment with a therapy disclosed herein, e.g., from Table2. In one embodiment, the subject, e.g., a patient, is assigned to asecond class if the mutation is not present. For example, a patient whohas a tumor that does not contain a fusion, may be determined as notbeing a candidate to receive a therapy disclosed herein, e.g., fromTable 2.

In another embodiment, responsive to the determination of the presenceof the fusion, the subject is identified as likely to respond to atreatment that comprises a therapy disclosed herein, e.g., from Table 2.

In yet another embodiment, responsive to the determination of thepresence of the fusion, the method includes administering a kinaseinhibitor, e.g., a kinase inhibitor as described herein, to the subject.

Method of Evaluating a Tumor or a Subject

In another aspect, the invention features a method of evaluating asubject (e.g., a patient), e.g., for risk of having or developing acancer, e.g., cholangiocarcinoma, e.g., a intrahepaticcholangiocarcinoma (ICC). The method includes: acquiring information orknowledge of the presence of a fusion as described herein in a subject(e.g., acquiring genotype information of the subject that identifies afusion as being present in the subject); acquiring a sequence for anucleic acid molecule identified herein (e.g., a nucleic acid moleculethat includes a fusion sequence); or detecting the presence of a fusionnucleic acid or polypeptide in the subject), wherein the presence of thefusion is positively correlated with increased risk for, or having, acancer associated with such a fusion.

The method can further include acquiring, e.g., directly or indirectly,a sample from a patient and evaluating the sample for the present of afusion as described herein.

The method can further include the step(s) of identifying (e.g.,evaluating, diagnosing, screening, and/or selecting) the subject asbeing positively correlated with increased risk for, or having, a cancerassociated with the fusion.

In another embodiment, a subject identified has having a fusion isidentified or selected as likely or unlikely to respond to a treatment,e.g., a therapy disclosed herein, e.g., from Table 2. The method canfurther include treating the subject with a therapy disclosed herein,e.g., from Table 2.

In a related aspect, a method of evaluating a patient or a patientpopulation is provided. The method includes: identifying, selecting, orobtaining information or knowledge that the patient or patientpopulation has participated in a clinical trial; acquiring informationor knowledge of the presence of a fusion in the patient or patientpopulation (e.g., acquiring genotype information of the subject thatidentifies a fusion as being present in the subject); acquiring asequence for a nucleic acid molecule identified herein (e.g., a nucleicacid molecule that includes a fusion sequence); or detecting thepresence of a fusion nucleic acid or polypeptide in the subject),wherein the presence of the fusion identifies the patient or patientpopulation as having an increased risk for, or having, acholangiocarcinoma associated with the fusion.

In some embodiments, the method further includes treating the subjectwith an inhibitor, e.g., a kinase inhibitor as described herein.

Reporting

Methods described herein can include providing a report, such as, inelectronic, web-based, or paper form, to the patient or to anotherperson or entity, e.g., a caregiver, e.g., a physician, e.g., anoncologist, a hospital, clinic, third-party payor, insurance company orgovernment office. The report can include output from the method, e.g.,the identification of nucleotide values, the indication of presence orabsence of a fusion as described herein, or wildtype sequence. In oneembodiment, a report is generated, such as in paper or electronic form,which identifies the presence or absence of an alteration describedherein, and optionally includes an identifier for the patient from whichthe sequence was obtained.

The report can also include information on the role of a fusion asdescribed herein, or wild-type sequence, in disease. Such informationcan include information on prognosis, resistance, or potential orsuggested therapeutic options. The report can include information on thelikely effectiveness of a therapeutic option, the acceptability of atherapeutic option, or the advisability of applying the therapeuticoption to a patient, e.g., a patient having a sequence, alteration ormutation identified in the test, and in embodiments, identified in thereport. For example, the report can include information, or arecommendation on, the administration of a drug, e.g., theadministration at a preselected dosage or in a preselected treatmentregimen, e.g., in combination with other drugs, to the patient. In anembodiment, not all mutations identified in the method are identified inthe report. For example, the report can be limited to mutations in geneshaving a preselected level of correlation with the occurrence,prognosis, stage, or susceptibility of the cancer to treatment, e.g.,with a preselected therapeutic option. The report can be delivered,e.g., to an entity described herein, within 7, 14, or 21 days fromreceipt of the sample by the entity practicing the method.

In another aspect, the invention features a method for generating areport, e.g., a personalized cancer treatment report, by obtaining asample, e.g., a tumor sample, from a subject, detecting a fusion asdescribed herein in the sample, and selecting a treatment based on themutation identified. In one embodiment, a report is generated thatannotates the selected treatment, or that lists, e.g., in order ofpreference, two or more treatment options based on the mutationidentified. In another embodiment, the subject, e.g., a patient, isfurther administered the selected method of treatment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, and theexample are illustrative only and not intended to be limiting.

The details of one or more embodiments featured in the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages featured in the invention will beapparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1C are tables summarizing the fusion molecules and therearrangement events described herein.

FIG. 1A summarizes the following: the name of the fusion (referred to as“fusion”); the tissue source (referred to as “disease”); the approximatelocations of the first and second breakpoints that give rise to therearrangement events (±50 nucleotides) (referred to as “Breakpoint 1”and “Breakpoint 2,” respectively); and the type of rearrangement(referred to as “rearrangement”).

FIG. 1B summarizes the following: the name of the fusion (referred to as“fusion”); the accession number of the full length sequences thatcontain the 5′- and the 3′-exon sequences (referred to as “5′ TranscriptID” and “3′ Transcript ID,” respectively); and the identity of theexon(s) of the 5′ transcript and the exon(s) of the 3′ transcript. Thesequences corresponding to the accession numbers provided in FIG. 1B areset forth in the figures appended herein. Alternatively, the sequencescan be found by searching the RefSeq Gene as databased at UCSC GenomeBrowser (genome.ucsc.edu). For example, the following link can be used:http://genome.ucsc.edu/cgi-bin/hgc?hgsid=359255927&c=chr10&o=123237843&t=123356159&g=refGene&i=NM_001144915to search for Accession Number=NM_001144915.

FIG. 1C summarizes the following: the name of the fusion; the SEQ IDNOs. of the 5′ partner and the 3′ partner; and the figure in which thesequence is shown. For example, the Nt and Aa sequences of FGFR2 haveSEQ ID NOs: 1 and 2, respectively, which are shown in FIGS. 2 and 3 ,respectively. The Nt and Aa sequences of TACC3 have SEQ ID NOs: 3 and 4,which are shown in FIGS. 4 and 5 , respectively.

FIGS. 2A-2B depict the nucleotide sequence of FGFR2 cDNA (NM_001144915,SEQ ID NO: 1). The exon boundaries are shown in bold and underlined. Thestart of the first exon and the end of the last exon are shown by asingle underline (e.g., shown as A). Further exons (second, third,fourth and so on) are indicated consecutively from 5′ to 3′ orientationby the underline of two consecutive nucleotides. For example,nucleotides GT at positions 169-170 correspond to the 3′-end of thefirst exon at position G, and the 5′-start of the second exon is atposition T. The start codon is shown in bold and italics. The stop codonis shown in italics and underlined.

FIG. 3 depicts the amino acid sequence of FGFR2 (SEQ ID NO: 2).

FIGS. 4A-4B depict the nucleotide sequence of TACC3 cDNA (NM_006342, SEQID NO: 3). The exon boundaries are shown in bold and underlined. Thestart of the first exon is shown by a single underline. Further exons(second, third, fourth) are indicated consecutively from 5′ to 3′orientation by the underline of two consecutive nucleotides (asexemplified in FIGS. 2A-2B above). The start codon is shown in bold anditalics. The stop codon is shown in italics and underlined.

FIG. 5 depicts the amino acid sequence of TACC3 (SEQ ID NO: 4).

FIGS. 6A-6C depict the nucleotide sequence of KIAA1598 cDNA(NM_001127211, SEQ ID NO: 5). The exon boundaries are shown in bold andunderlined. The start of the first exon is shown by a single underline.Further exons (second, third, fourth) are indicated consecutively from5′ to 3′ orientation by the underline of two consecutive nucleotides (asexemplified in FIGS. 2A-2B above). The start codon is shown in bold anditalics. The stop codon is shown in italics and underlined.

FIG. 7 depicts the amino acid sequence of KIAA1598 (SEQ ID NO: 6).

FIGS. 8A-8B depict the nucleotide sequence of BICC1 cDNA (NM_001080512,SEQ ID NO: 7). The exon boundaries are shown in bold and underlined. Thestart of the first exon is shown by a single underline. Further exons(second, third, fourth) are indicated consecutively from 5′ to 3′orientation by the underline of two consecutive nucleotides (asexemplified in FIGS. 2A-2B above). The start codon is shown in bold anditalics. The stop codon is shown in italics and underlined.

FIG. 9 depicts the amino acid sequence of BICC1 (SEQ ID NO: 8).

FIGS. 10A-10B depict the nucleotide sequence of PARK2 cDNA (NM_004562,SEQ ID NO: 9). The exon boundaries are shown in bold and underlined. Thestart of the first exon is shown by a single underline. Further exons(second, third, fourth) are indicated consecutively from 5′ to 3′orientation by the underline of two consecutive nucleotides (asexemplified in FIGS. 2A-2B above). The start codon is shown in bold anditalics. The stop codon is shown in italics and underlined.

FIG. 11 depicts the amino acid sequence of PARK2 (SEQ ID NO: 10).

FIGS. 12A-12B depict the nucleotide sequence of FGFR2 cDNA (NM_000141,SEQ ID NO: 11). The exon boundaries are shown in bold and underlined.The start of the first exon is shown by a single underline. Furtherexons (second, third, fourth) are indicated consecutively from 5′ to 3′orientation by the underline of two consecutive nucleotides (asexemplified in FIGS. 2A-2B above). The start codon is shown in bold anditalics. The stop codon is shown in italics and underlined.

FIG. 13 depicts the amino acid sequence of FGFR2 (SEQ ID NO: 12).

FIGS. 14A-14B depict the nucleotide sequence of NOL4 cDNA (NM_003787,SEQ ID NO: 13). The exon boundaries are shown in bold and underlined.The start of the first exon is shown by a single underline. Furtherexons (second, third, fourth) are indicated consecutively from 5′ to 3′orientation by the underline of two consecutive nucleotides (asexemplified in FIGS. 2A-2B above). The start codon is shown in bold anditalics. The stop codon is shown in italics and underlined.

FIG. 15 depicts the amino acid sequence of NOL4 (SEQ ID NO: 14).

FIG. 16 depicts the nucleotide sequence of ZDHHC6 cDNA (NM_022494, SEQID NO: 15). The exon boundaries are shown in bold and underlined. Thestart of the first exon is shown by a single underline. Further exons(second, third, fourth) are indicated consecutively from 5′ to 3′orientation by the underline of two consecutive nucleotides (asexemplified in FIGS. 2A-2B above). The start codon is shown in bold anditalics. The stop codon is shown in italics and underlined.

FIG. 17 depicts the amino acid sequence of ZDHHC6 (SEQ ID NO: 16).

DETAILED DESCRIPTION

Described herein are novel alterations, e.g., rearrangement events,found in cholangiocarcinomas. In certain embodiments, the rearrangementevents are found in an FGFR2 gene or an NTRK gene, e.g., as exemplifiedin Table 1, FIGS. 1A-1C and FIGS. 2-17 . In certain embodiments, thenovel rearrangement events give rise to fusion molecules that includes afragment of a first gene and a fragment of a second gene, e.g., a fusionthat includes a 5′-exon and a 3′-exon summarized in FIGS. 1A-1C andFIGS. 2-17 . The term “fusion” or “fusion molecule” is used genericallyherein, and includes any fusion molecule (e.g., gene, gene product(e.g., cDNA, mRNA, or polypeptide), and variant thereof) that includes afragment of first gene and a fragment of second gene described herein,including, e.g., an FGFR2-TACC3, FGFR2-KIAA1598, BICC1-FGFR2,FGFR2-BICC1, PARK2-FGFR2, FGFR2-NOL4, ZDHHC6-FGFR2, or RABGAP1L-NTRK1,e.g., as described in Table 1, FIGS. 1A-1C and FIGS. 2-17 . Expressionof the fusion molecules was detected in cholangiocarcinomas, thussuggesting an association with neoplastic growth or cancer (includingpre-malignant, or malignant and/or metastatic growth).

Cholangiocarcinoma (also known as bile duct cancer) can arise from thetissues in the bile duct. Cholangiocarcinoma can occur in any part ofthe bile duct. The part of the tube that is outside of the liver iscalled extrahepatic. It is in this portion of the bile duct where cancerusually arises. A perihilar cancer, also called a Klatskin tumor, beginswhere many small channels join into the bile duct at the point where itleaves the liver. About two-thirds of all cholangiocarcinomas occurhere. Distal cholangiocarcinoma occurs at the opposite end of the ductfrom perihilar cancer, near where the bile duct empties into the smallintestine. About one-fourth of all cholangiocarcinomas are distalcholangiocarcinomas. About 5% to 10% of cholangiocarcinomas areintrahepatic, or inside the liver. Adenocarcinoma is the most commontype of extrahepatic cholangiocarcinoma, and accounting for up to 95% ofall cholangiocarcinomas. Adenocarcinoma is cancer arising from the mucusglands lining the inside of the bile duct. Cholangiocarcinoma is anotherterm that may be used to describe this type of cancer.

Accordingly, the invention provides, at least in part, the following:methods for treating a cholangiocarcinoma using an inhibitor of one ofthe alterations described herein, e.g., an FGFR2 or an NTRK1 inhibitor;methods for identifying, assessing or detecting an alteration, e.g.,fusion molecule as described herein; methods for identifying, assessing,evaluating, and/or treating a subject having a cancer, e.g., acholangiocarcinoma having a fusion molecule as described herein;isolated fusion nucleic acid molecules, nucleic acid constructs, hostcells containing the nucleic acid molecules; purified fusionpolypeptides and binding agents; detection reagents (e.g., probes,primers, antibodies, kits, capable, e.g., of specific detection of afusion nucleic acid or protein); screening assays for identifyingmolecules that interact with, e.g., inhibit, the fusions, e.g., novelkinase inhibitors; as well as assays and kits for evaluating,identifying, assessing and/or treating a subject having a cancer, e.g.,a cholangiocarcinoma having a fusion. The compositions and methodsidentified herein can be used, for example, to identify new inhibitors;to evaluate, identify or select a subject, e.g., a patient, having acancer; and to treat or prevent a cancer, such as a cholangiocarcinoma.

Certain terms are defined. Additional terms are defined throughout thespecification.

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

“Acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a physical entity, or a value, e.g., a numericalvalue, by “directly acquiring” or “indirectly acquiring” the physicalentity or value. “Directly acquiring” means performing a process (e.g.,performing a synthetic or analytical method) to obtain the physicalentity or value. “Indirectly acquiring” refers to receiving the physicalentity or value from another party or source (e.g., a third partylaboratory that directly acquired the physical entity or value).Directly acquiring a physical entity includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial. Exemplary changes include making a physical entity from two ormore starting materials, shearing or fragmenting a substance, separatingor purifying a substance, combining two or more separate entities into amixture, performing a chemical reaction that includes breaking orforming a covalent or non-covalent bond. Directly acquiring a valueincludes performing a process that includes a physical change in asample or another substance, e.g., performing an analytical processwhich includes a physical change in a substance, e.g., a sample,analyte, or reagent (sometimes referred to herein as “physicalanalysis”), performing an analytical method, e.g., a method whichincludes one or more of the following: separating or purifying asubstance, e.g., an analyte, or a fragment or other derivative thereof,from another substance; combining an analyte, or fragment or otherderivative thereof, with another substance, e.g., a buffer, solvent, orreactant; or changing the structure of an analyte, or a fragment orother derivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.

“Acquiring a sequence” as the term is used herein, refers to obtainingpossession of a nucleotide sequence or amino acid sequence, by “directlyacquiring” or “indirectly acquiring” the sequence. “Directly acquiring asequence” means performing a process (e.g., performing a synthetic oranalytical method) to obtain the sequence, such as performing asequencing method (e.g., a Next Generation Sequencing (NGS) method).“Indirectly acquiring a sequence” refers to receiving information orknowledge of, or receiving, the sequence from another party or source(e.g., a third party laboratory that directly acquired the sequence).The sequence acquired need not be a full sequence, e.g., sequencing ofat least one nucleotide, or obtaining information or knowledge, thatidentifies a fusion disclosed herein as being present in a subjectconstitutes acquiring a sequence.

Directly acquiring a sequence includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial, such as a tissue sample, e.g., a biopsy, or an isolatednucleic acid (e.g., DNA or RNA) sample. Exemplary changes include makinga physical entity from two or more starting materials, shearing orfragmenting a substance, such as a genomic DNA fragment; separating orpurifying a substance (e.g., isolating a nucleic acid sample from atissue); combining two or more separate entities into a mixture,performing a chemical reaction that includes breaking or forming acovalent or non-covalent bond. Directly acquiring a value includesperforming a process that includes a physical change in a sample oranother substance as described above.

“Acquiring a sample” as the term is used herein, refers to obtainingpossession of a sample, e.g., a tissue sample or nucleic acid sample, by“directly acquiring” or “indirectly acquiring” the sample. “Directlyacquiring a sample” means performing a process (e.g., performing aphysical method such as a surgery or extraction) to obtain the sample.“Indirectly acquiring a sample” refers to receiving the sample fromanother party or source (e.g., a third party laboratory that directlyacquired the sample). Directly acquiring a sample includes performing aprocess that includes a physical change in a physical substance, e.g., astarting material, such as a tissue, e.g., a tissue in a human patientor a tissue that has was previously isolated from a patient. Exemplarychanges include making a physical entity from a starting material,dissecting or scraping a tissue; separating or purifying a substance(e.g., a sample tissue or a nucleic acid sample); combining two or moreseparate entities into a mixture; performing a chemical reaction thatincludes breaking or forming a covalent or non-covalent bond. Directlyacquiring a sample includes performing a process that includes aphysical change in a sample or another substance, e.g., as describedabove.

“Binding entity” means any molecule to which molecular tags can bedirectly or indirectly attached that is capable of specifically bindingto an analyte. The binding entity can be an affinity tag on a nucleicacid sequence. In certain embodiments, the binding entity allows forseparation of the nucleic acid from a mixture, such as an avidinmolecule, or an antibody that binds to the hapten or an antigen-bindingfragment thereof. Exemplary binding entities include, but are notlimited to, a biotin molecule, a hapten, an antibody, an antibodybinding fragment, a peptide, and a protein.

“Complementary” refers to sequence complementarity between regions oftwo nucleic acid strands or between two regions of the same nucleic acidstrand. It is known that an adenine residue of a first nucleic acidregion is capable of forming specific hydrogen bonds (“base pairing”)with a residue of a second nucleic acid region which is antiparallel tothe first region if the residue is thymine or uracil. Similarly, it isknown that a cytosine residue of a first nucleic acid strand is capableof base pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.In certain embodiments, the first region comprises a first portion andthe second region comprises a second portion, whereby, when the firstand second portions are arranged in an antiparallel fashion, at leastabout 50%, at least about 75%, at least about 90%, or at least about 95%of the nucleotide residues of the first portion are capable of basepairing with nucleotide residues in the second portion. In otherembodiments, all nucleotide residues of the first portion are capable ofbase pairing with nucleotide residues in the second portion.

The term “cancer” or “tumor” is used interchangeably herein. These termsrefer to the presence of cells possessing characteristics typical ofcancer-causing cells, such as uncontrolled proliferation, immortality,metastatic potential, rapid growth and proliferation rate, and certaincharacteristic morphological features. In one embodiment, the cancer isa cholangiocarcinoma.

The term “neoplasm” or “neoplastic” cell refers to an abnormalproliferative stage, e.g., a hyperproliferative stage, in a cell ortissue that can include a benign, pre-malignant, malignant (cancer) ormetastatic stage.

Cancer is “inhibited” if at least one symptom of the cancer isalleviated, terminated, slowed, or prevented. As used herein, cancer isalso “inhibited” if recurrence or metastasis of the cancer is reduced,slowed, delayed, or prevented.

“Chemotherapeutic agent” means a chemical substance, such as a cytotoxicor cytostatic agent, that is used to treat a condition, particularlycancer.

As used herein, “cancer therapy” and “cancer treatment” are synonymousterms.

As used herein, “chemotherapy” and “chemotherapeutic” and“chemotherapeutic agent” are synonymous terms.

The terms “homology” or “identity,” as used interchangeably herein,refer to sequence similarity between two polynucleotide sequences orbetween two polypeptide sequences, with identity being a more strictcomparison. The phrases “percent identity or homology” and “% identityor homology” refer to the percentage of sequence similarity found in acomparison of two or more polynucleotide sequences or two or morepolypeptide sequences. “Sequence similarity” refers to the percentsimilarity in base pair sequence (as determined by any suitable method)between two or more polynucleotide sequences. Two or more sequences canbe anywhere from 0-100% similar, or any integer value there between.Identity or similarity can be determined by comparing a position in eachsequence that can be aligned for purposes of comparison. When a positionin the compared sequence is occupied by the same nucleotide base oramino acid, then the molecules are identical at that position. A degreeof similarity or identity between polynucleotide sequences is a functionof the number of identical or matching nucleotides at positions sharedby the polynucleotide sequences. A degree of identity of polypeptidesequences is a function of the number of identical amino acids atpositions shared by the polypeptide sequences. A degree of homology orsimilarity of polypeptide sequences is a function of the number of aminoacids at positions shared by the polypeptide sequences. The term“substantially identical,” as used herein, refers to an identity orhomology of at least 75%, at least 80%, at least 85%, at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

“Likely to” or “increased likelihood,” as used herein, refers to anincreased probability that an item, object, thing or person will occur.Thus, in one example, a subject that is likely to respond to treatmentwith a kinase inhibitor, alone or in combination, has an increasedprobability of responding to treatment with the inhibitor alone or incombination, relative to a reference subject or group of subjects.

“Unlikely to” refers to a decreased probability that an event, item,object, thing or person will occur with respect to a reference. Thus, asubject that is unlikely to respond to treatment with a kinaseinhibitor, alone or in combination, has a decreased probability ofresponding to treatment with a kinase inhibitor, alone or incombination, relative to a reference subject or group of subjects.

“Sequencing” a nucleic acid molecule requires determining the identityof at least 1 nucleotide in the molecule. In embodiments, the identityof less than all of the nucleotides in a molecule are determined. Inother embodiments, the identity of a majority or all of the nucleotidesin the molecule is determined.

“Next-generation sequencing or NGS or NG sequencing” as used herein,refers to any sequencing method that determines the nucleotide sequenceof either individual nucleic acid molecules (e.g., in single moleculesequencing) or clonally expanded proxies for individual nucleic acidmolecules in a highly parallel fashion (e.g., greater than 10⁵ moleculesare sequenced simultaneously). In one embodiment, the relative abundanceof the nucleic acid species in the library can be estimated by countingthe relative number of occurrences of their cognate sequences in thedata generated by the sequencing experiment. Next generation sequencingmethods are known in the art, and are described, e.g., in Metzker, M.(2010) Nature Biotechnology Reviews 11:31-46, incorporated herein byreference. Next generation sequencing can detect a variant present inless than 5% of the nucleic acids in a sample.

“Sample,” “tissue sample,” “patient sample,” “patient cell or tissuesample” or “specimen” each refers to a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissuesample can be solid tissue as from a fresh, frozen and/or preservedorgan, tissue sample, biopsy, or aspirate; blood or any bloodconstituents; bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid or interstitial fluid; or cells from any time ingestation or development of the subject. The tissue sample can containcompounds that are not naturally intermixed with the tissue in naturesuch as preservatives, anticoagulants, buffers, fixatives, nutrients,antibiotics or the like. In one embodiment, the sample is preserved as afrozen sample or as formaldehyde- or paraformaldehyde-fixedparaffin-embedded (FFPE) tissue preparation. For example, the sample canbe embedded in a matrix, e.g., an FFPE block or a frozen sample.

A “tumor nucleic acid sample” as used herein, refers to nucleic acidmolecules from a tumor or cancer sample. Typically, it is DNA, e.g.,genomic DNA, or cDNA derived from RNA, from a tumor or cancer sample. Incertain embodiments, the tumor nucleic acid sample is purified orisolated (e.g., it is removed from its natural state).

A “control” or “reference” “nucleic acid sample” as used herein, refersto nucleic acid molecules from a control or reference sample. Typically,it is DNA, e.g., genomic DNA, or cDNA derived from RNA, not containingthe alteration or variation in the gene or gene product, e.g., notcontaining a fusion. In certain embodiments, the reference or controlnucleic acid sample is a wild type or a non-mutated sequence. In certainembodiments, the reference nucleic acid sample is purified or isolated(e.g., it is removed from its natural state). In other embodiments, thereference nucleic acid sample is from a non-tumor sample, e.g., a bloodcontrol, a normal adjacent tumor (NAT), or any other non-canceroussample from the same or a different subject.

“Adjacent to the interrogation position,” as used herein, means that asite sufficiently close such that a detection reagent complementary withthe site can be used to distinguish between a mutation, e.g., a mutationdescribed herein, and a reference sequence, e.g., a non-mutant orwild-type sequence, in a target nucleic acid. Directly adjacent, as usedherein, is where 2 nucleotides have no intervening nucleotides betweenthem.

“Associated mutation,” as used herein, refers to a mutation within apreselected distance, in terms of nucleotide or primary amino acidsequence, from a definitional mutation, e.g., a mutant as describedherein, e.g., a translocation, breakpoint or fusion molecule describedherein. In embodiments, the associated mutation is within n, wherein nis 2, 5, 10, 20, 30, 50, 100, or 200 nucleotides from the definitionalmutation (n does not include the nucleotides defining the associated anddefinitional mutations). In embodiments, the associated mutation is atranslocation mutation.

“Interrogation position,” as used herein, comprises at least onenucleotide (or, in the case of polypeptides, an amino acid residue)which corresponds to a nucleotide (or amino acid residue) that ismutated in a mutation of interest, e.g., a mutation being identified, orin a nucleic acid (or protein) being analyzed, e.g., sequenced, orrecovered.

A “reference sequence,” as used herein, e.g., as a comparator for amutant sequence, is a sequence which has a different nucleotide or aminoacid at an interrogation position than does the mutant(s) beinganalyzed. In an embodiment, the reference sequence is wild-type for atleast the interrogation position.

Headings, e.g., (a), (b), (i) etc, are presented merely for ease ofreading the specification and claims. The use of headings in thespecification or claims does not require the steps or elements beperformed in alphabetical or numerical order or the order in which theyare presented.

Various aspects featured in the invention are described in furtherdetail below. Additional definitions are set out throughout thespecification.

FGFR2 and NTRK1 Alterations

Described herein are novel rearrangements of the FGFR2 and NTRK1 genesin cholangiocarcinomas.

FGFR2 Alterations

The FGFR family plays an important role in cell differentiation, growthand angiogenesis (reviewed in Powers et al. (2000), Endocr. Relat.Cancer, 7(3):165-197, and gain of function mutations in FGFRs have beenreported in several cancer types (reviewed in Eswarakumar et al. (2005),Cytokine Growth Factor Rev., 16(2):139-149).

FGFR2 (Fibroblast growth factor receptor 2) is a member of thefibroblast growth factor receptor family, where amino acid sequence ishighly conserved between members and throughout evolution. FGFR familymembers differ from one another in their ligand affinities and tissuedistribution. A full-length representative protein consists of anextracellular region, composed of three immunoglobulin-like domains, asingle hydrophobic membrane-spanning segment and a cytoplasmic tyrosinekinase domain. FGFR2 is composed of three immunoglobulin c-2 typedomains, one transmembrane domain, and one tyrosine kinase catalyticdomain. The extracellular portion of the protein interacts withfibroblast growth factors, setting in motion a cascade of downstreamsignals, ultimately influencing mitogenesis and differentiation. Thisparticular family member is a high-affinity receptor for acidic, basicand/or keratinocyte growth factor, depending on the isoform. Multiplealternatively spliced transcript variants encoding different isoformshave been noted for the FGFR2 gene. The FGFR2 amino and nucleotidesequences are known in the art. Exemplary sequences for human FGFR2 areprovided herein as SEQ ID NOs:1 and 11, and FIGS. 2 and 12 (nucleotide),and SEQ ID NOs:2 and 12, and FIGS. 3 and 13 (amino acid).

FGFR2 amplification has been reported in several cancer types, mostfrequently in gastric cancer (3-4%) (Matsumoto et al., 2012, Br. J.Cancer, 106(4):727-732, Hara et al., 1998, Lab Invest., 78(9):1143-1153)and breast cancer (1-11%) (Heiskanen et al., 2001, Anal Cell Pathol.22(4):229-234, Adnane et al., 1991; Oncogene 6(4):659-663, Turner etal., 2010, Oncogene 29(14):2013-2023). FGFR2 has been shown to beexpressed in cholangiocarcinoma, leading to activation of the MEK1/2pathway (Narong et al., 2011, Oncol. Lett. 2(5):821-825). The FGFR2alterations described herein are expected to result in activation and/orupregulation of the FGFR2 protein. Accordingly, treatment with an agentthat reduces (e.g., inhibits) FGFR2 is encompassed by the invention. Inone embodiment, the agent is Regorafenib. Regorafenib inhibits cellularkinases including FGFR2, and has been approved for treatment of somemetastatic colorectal cancer (mCRC) patients (FDA.gov, November 2012).The multi-kinase inhibitor ponatinib (AP24534), recently approved by theFDA for use in chronic myelogenous leukemia based on the results of aPhase 2 trial, has also been shown in preclinical studies to havesubstantial activity against all four FGFR kinases (Cortes et al., 2012,American Society of Hematology ASH, Abstract 163, Gozgit et al., 2012,Mol. Cancer Ther., 11(3):690-699). Clinical trials of multiple Fgfrinhibitors are currently underway (Turner and Grose, 2010, Nat. Rev.Cancer, 10(2):116-129).

Each of the FGFR2 alterations is described herein in more detail

FGFR2-TACC3

The FGFR2-TACC3 fusion has not been reported. However, similarFGFR3-TACC3 fusions have been previously reported in glioblastoma and ina bladder cancer cell line; these fusions were found to be activatingand to have transformative potential (Williams et al., Hum. Mol. GenetePub, December 2012, Singh et al., 2012, Science 337(6099):1231-1235).The FGFR2-TACC3 fusion is therefore expected to be oncogenic. FGFR2amplification has also been reported in several cancer types, mostfrequently in gastric cancer and breast cancer as described herein.Inhibitors of FGFR2, such as Regorafenib and ponatinib can be used totreat cholangiosarcoma.

In one embodiment, the rearrangement, nucleotide and amino acidsequences for FGFR2 (exons 1-16)-TACC3 (exons 11-16) are depicted inFIGS. 1A-5 and SEQ ID NOs. 1-4.

FGFR2-KIAA1598

The FGFR2-KIAA1598 rearrangement results in truncation of the 3′UTR ofthe FGFR2 gene, which can result in upregulation of the FGFR2 protein.FGFR2 amplification has also been reported in several cancer types, mostfrequently in gastric cancer and breast cancer as described herein.Inhibitors of FGFR2, such as Regorafenib and ponatinib can be used totreat cholangiosarcoma.

In one embodiment, the rearrangement, nucleotide and amino acidsequences for FGFR2 (exons 1-16)-KIAA1598 (exons 7-17) are depicted inFIGS. 1A-1C, 2-3 and 6-7 and SEQ ID NOs. 1-2 and 5-6.

BICC1-FGFR2

The BICC1-FGFR2 fusion has not been reported in cholangiocarcinoma, orother cancers. FGFR2 amplification has also been reported in severalcancer types, most frequently in gastric cancer and breast cancer asdescribed herein. Treatment Inhibitors of FGFR2, such as Regorafenib andponatinib can be used to treat cholangiosarcoma.

In one embodiment, the rearrangement, nucleotide and amino acidsequences for BICC1 (exons 1-2)-FGFR2 (exon 17) are depicted in FIGS.1A-1C, 2-3 and 8-9 and SEQ ID NOs. 1-2 and 7-8.

FGFR2-BICC1

The FGFR2-BICC1 result in an in-frame fusion including the N-terminalportion of FGFR2 (containing the kinase domain) nearly the entire codingsequence of BICC1 (García-Mayoral et al., 2007, Structure 15(4):485-498,Kim and Bowie, 2003, Trends Biochem. Sci. 28(12):625-628). Otherin-frame fusions containing the kinase domain of FGFR2 have been shownto result in kinase activation (Singh et al., 2012, Science337(6099):1231-1235, Lorenzi et al., 1996, Proc. Natl. Acad. Sci. USA,93(17):8956-8961). A recent report has described an FGFR2 fusion gene incholangiocarcinoma (Wu et al. Cancer Discov ePub, May 2013). FGFR2mutations have been reported in 2% of tumors analyzed in COSMIC, withthe highest prevalence in endometrial cancer (10%) and lower incidencein several other cancers (COSMIC, February 2013). F GFR2 signaling hasbeen described as tumorigenic in lung, pancreatic and gastric cancers(Yamayoshi et al., 2004, J. Pathol., 204(1):110-118; Cho et al., 2007,Am. J. Pathol., 170(6):1964-1974; Toyokawa et al., 2009, Oncol. Rep.,21(4):875-880). However, FGFR2 has also been described as a tumorsuppressor in the context of other cancers, such as melanoma (Gartsideet al., 2009, Mol. Cancer Res., 7(1):41-54). Clinical trials of multipleFGFR inhibitors are currently underway (Turner and Grose, 2010,Oncogene, 29(14):2013-2023). Inhibitors of FGFR2, such as Regorafeniband ponatinib can be used to treat cholangiosarcoma.

In one embodiment, the rearrangement, nucleotide and amino acidsequences for FGFR2 (exons 1-16)-BICC1 (exons 18-21) are depicted inFIGS. 1A-1C, 2-3 and 8-9 and SEQ ID NOs. 1-2 and 7-8.

PARK2-FGFR2

The PARK2-FGFR2 fusion results in a fusion that includes the N-terminalportion of PARK2, which encodes the E3 ligase parkin, and the last exon(aa 768-821) of FGFR2 (Uniprot). The portion of FGFR2 not included inthis fusion is predicted to encode a protein truncated after thefunctional kinase domain. Similar truncations of FGFR2 (764* and 776*)have been described as oncogenic, efficiently transforming culturedcells (Lorenzi et al., 1997, Oncogene 15(7):817-26). Therefore, thisfusion is expected to activate the FGFR2 signaling.

FGFR2 amplification has also been reported in several cancer types, mostfrequently in gastric cancer and breast cancer as described herein.Treatment Inhibitors of FGFR2, such as Regorafenib and ponatinib can beused to treat cholangiosarcoma.

In one embodiment, the rearrangement, nucleotide and amino acidsequences for PARK2 (exons 1-9)-FGFR2 (exon 18) are depicted in FIGS.1A-1C and 10-13 and SEQ ID NOs. 9-12.

In one embodiment, the rearrangement comprises a fusion of PARK2(intron9) to FGFR2 (intron17). The expected genomic coordinates are:

-   -   FGFR2 breakpoint: chr10:123239535-123243212.    -   PARK2 breakpoint: chr6:161807909-161969886.

The fusion is comprised of 10 complete exons, all coming from thereverse strand. The fusion is in frame. The orientation of the fusion isexpected to be 5′ fusion partner exons: PARK2 (exons1-9) to 3′ fusionpartner exons: FGFR2 (exon18).

The fused domains include:

-   -   (i) PARK2, E3 ubiquitin-protein ligase parkin, has one ubiquitin        homologue domain and two zink finger domains. The fusion, which        includes exons 1-9 of PARK2 contains the entire ubiquitin        homologue domain and part of the first zink finger domain, which        are the core set of exons to give reasonable activity; and (ii)        FGFR2, the fusion includes the last exon of FGFR2.

The refSeq IDs for the nucleotide and amino acid sequences are:

-   -   PARK2: NM_004562 provided herein as SEQ ID NOs: 9-10 and FIGS.        10-11 , respectively.    -   FGFR2: NM_000141 provided herein as SEQ ID NOs: 11-12 and FIGS.        12-13 , respectively.

FGFR2-NOL4

The FGFR2-NOL4 fusion results in an in-frame fusion, containingtranscribed exons 1-17 of FGFR2 (coding for amino acids 1-768) fused toNOL4 transcribed exons 7-11 (coding for amino acids 353-638). Theresulting fusion protein contains the N-terminus of FGFR2, whichincludes the protein kinase domain, fused to the C-terminus of the NOL4protein (UniProt.org). FGFR2-involving fusions containing the FGFR2kinase domain have been reported to be activating and oncogenic,including FGFR-TACC and FGFR2-FRAG1 (Singh et al., 2012, Science337(6099):1231-1235, Lorenzi et al., 1996, Proc. Natl. Acad. Sci. USA,93(17):8956-8961). FGFR2 mRNA has been shown to be expressed incholangiocarcinoma cell lines, leading to activation of the MEK1/2pathway (Narong and Leelawat, 2011, supra). Tumors with FGFR2amplification or activating mutations can be sensitive to FGFRinhibitors as described herein. FGFR2 has been associated withresistance to chemotherapeutics; shRNA inhibition of FGFR2 increased thesensitivity of ovarian epithelial cancer cells to cisplatin (Cole etal., 2010, Cancer Biol Ther 10(5):495-504). Inhibitors of FGFR2, such asRegorafenib and ponatinib can be used to treat cholangiosarcoma.

In one embodiment, the rearrangement, nucleotide and amino acidsequences for FGFR2 (exons 1-17) and NOL4 (exons 7-11) are depicted inFIGS. 1A-1C and 12-15 and SEQ ID NOs. 11-14.

In one embodiment, the rearrangement comprises a fusion of FGFR2(intronl7) to NOL4 (intron 6). The expected genomic coordinates are:

-   -   FGFR2 breakpoint: chr10:123239535-123243212.    -   NOL4 breakpoint: chr18:31538203-31599282.

The fusion is comprised of 22 complete exons, all coming from thereverse strand. The fusion is in frame.

The orientation of the fusion is expected to be 5′ fusion partner exons:FGFR2 (exons1-17) to 3′ fusion partner exons: NOL4 (exons7-11).

The fused domains include:

FGFR2, the fusion includes the core set of exons for all active domainsof this transmembrane protein.

The refSeq IDs for the nucleotide and amino acid sequences are:

-   -   FGFR2: NM_000141 provided herein as SEQ ID NOs: 11-12 and FIGS.        12-13 , respectively.    -   NOL4: NM_003787 provided herein as SEQ ID NOs: 13-14 and FIGS.        14-15 , respectively.

ZDHHC6-FGFR2

The ZDHHC6-FGFR2 fusion results in a fusion including the N-terminalportion of ZDHHC6 (exons 1-5), which encodes the integral transmembranedomain of a palmitoyltransferase ZDHHC6, and the last exon (aa 768-821)of FGFR2 (Uniprot). The portion of FGFR2 not included in this fusion ispredicted to encode a protein truncated after the functional kinasedomain. Similar truncations of FGFR2 (764* and 776*) have been describedas oncogenic, efficiently transforming cultured cells (Lorenzi et al.,1997, Oncogene 15(7):817-26). Therefore, the ZDHHC6-FGFR2 fusion ispredicted to activate FGFR2 signaling by truncating the remaining FGFR2allele. A recent report has described an FGFR2 fusion gene incholangiocarcinoma, as well as a truncated FGFR2 similar to the oneobserved here in a patient with prostate cancer (Wu et al. Cancer DiscovePub, May 2013). Inhibitors of FGFR2, such as Regorafenib and ponatinibcan be used to treat cholangiosarcoma.

In one embodiment, the rearrangement, nucleotide and amino acidsequences for ZDHHC6 (exons 1-5) and FGFR2 (exon 18) are depicted inFIGS. 1A-1C and 12-13 and 16-17 and SEQ ID NOs. 11-12 and 15-16.

In one embodiment, the rearrangement comprises a fusion of ZDHHC6(intron5) to FGFR2 (intron17). The expected genomic coordinates are:

-   -   FGFR2 breakpoint: chr10:123239535-123243212    -   ZDHHC6 breakpoint: chr10:114198147-114200292

The fusion is comprised of 6 complete exons, all coming from the reversestrand. The fusion is in frame.

The orientation of the fusion is expected to be 5′ fusion partner exons:ZDHHC6 (exons1-5) to 3′ fusion partner exons: FGFR2 (exon18).

The fused domains include:

-   -   (i) ZDHHC6 is a zinc-finger involved in transferase activity,        transferring acyl groups and zinc ion binding. It contains 4        potential transmembrane domains and one zinc finger domain. All        of these domains are contained within the first 5 exons, and        therefore retained in the fusion product; and    -   (ii) FGFR2, the fusion includes the last exon of FGFR2.

The annotations above are based on the following refSeq IDs

-   -   ZDHHC6: NM_022494 provided herein as SEQ ID NOs: 15-16 and FIGS.        16-17 , respectively.    -   FGFR2: NM_000141 provided herein as SEQ ID NOs: 11-12 and FIGS.        12-13 , respectively.

RABGAP1L-NTRK1

NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1) is a member ofthe neurotrophic tyrosine kinase receptor (NTKR) family. This kinase isa membrane-bound receptor that, upon neurotrophin binding,phosphorylates itself and members of the MAPK pathway. The presence ofthis kinase leads to cell differentiation and may play a role inspecifying sensory neuron subtypes. Mutations in this gene have beenassociated with congenital insensitivity to pain, anhidrosis,self-mutilating behavior, mental retardation and cancer. Alternatetranscriptional splice variants of this gene have been found. The NTRK1amino and nucleotide sequences are known in the art. An exemplary aminoacid and nucleotide sequence for human NTRK1 are provided herein as SEQID NO:9 and SEQ ID NO:10, respectively.

NCBI Reference Sequence: NP_001012331 (SEQ ID NO: 9)    1mlrggrrgql gwhswaagpg sllawlilas agaapcpdac cphgssglrc trdgaldslh   61hlpgaenlte lyienqqhlq hlelrdlrgl gelrnltivk sglrfvapda fhftprlsrl  121nlsfnalesl swktvqglsl qelvlsgnpl hcscalrwlq rweeeglggv peqklqchgq  181gplahmpnas cgvptlkvqv pnasvdvgdd vllrcqvegr gleqagwilt eleqsatvmk  241sgglpslglt lanvtsdlnr knvtcwaend vgraevsvqv nvsfpasvql htavemhhwc  301ipfsvdgqpa pslrwlings vlnetsfift eflepaanet vrhgclrlnq pthvnngnyt  361llaanpfgqa sasimaafmd npfefnpedp ipdtnstsgd pvekkdetpf gvsvavglav  421faclflstll lvlnkcgrrn kfginrpavl apedglamsl hfmtlggssl sptegkgsgl  481qghiienpqy fsdacvhhik rrdivlkwel gegafgkvfl aechnllpeq dkmlvavkal  541keasesarqd fqreaelltm lqhqhivrff gvctegrpll mvfeymrhgd lnrflrshgp  601dakllagged vapgplglgq llavasqvaa gmvylaglhf vhrdlatrnc lvgqglvvki  661gdfgmsrdiy stdyyrvggr tmlpirwmpp esilyrkftt esdvwsfgvv lweiftygkq  721pwyqlsntea idcitqgrel erpracppev yaimrgcwqr epqqrhsikd vharlqalaq  781appvyldvlg Reference Sequence: NM_001012331 (SEQ ID NO: 10)    1tgcagctggg agcgcacaga cggctgcccc gcctgagcga ggcgggcgcc gccgcgatgc    61tgcgaggcgg acggcgcggg cagcttggct ggcacagctg ggctgcgggg ccgggcagcc  121tgctggcttg gctgatactg gcatctgcgg gcgccgcacc ctgccccgat gcctgctgcc  181cccacggctc ctcgggactg cgatgcaccc gggatggggc cctggatagc ctccaccacc  241tgcccggcgc agagaacctg actgagctct acatcgagaa ccagcagcat ctgcagcatc  301tggagctccg tgatctgagg ggcctggggg agctgagaaa cctcaccatc gtgaagagtg  361gtctccgttt cgtggcgcca gatgccttcc atttcactcc tcggctcagt cgcctgaatc  421tctccttcaa cgctctggag tctctctcct ggaaaactgt gcagggcctc tccttacagg  481aactggtcct gtcggggaac cctctgcact gttcttgtgc cctgcgctgg ctacagcgct  541gggaggagga gggactgggc ggagtgcctg aacagaagct gcagtgtcat gggcaagggc  601ccctggccca catgcccaat gccagctgtg gtgtgcccac gctgaaggtc caggtgccca  661atgcctcggt ggatgtgggg gacgacgtgc tgctgcggtg ccaggtggag gggcggggcc  721tggagcaggc cggctggatc ctcacagagc tggagcagtc agccacggtg atgaaatctg  781ggggtctgcc atccctgggg ctgaccctgg ccaatgtcac cagtgacctc aacaggaaga  841acgtgacgtg ctgggcagag aacgatgtgg gccgggcaga ggtctctgtt caggtcaacg  901tctccttccc ggccagtgtg cagctgcaca cggcggtgga gatgcaccac tggtgcatcc  961ccttctctgt ggatgggcag ccggcaccgt ctctgcgctg gctcttcaat ggctccgtgc 1021tcaatgagac cagcttcatc ttcactgagt tcctggagcc ggcagccaat gagaccgtgc 1081ggcacgggtg tctgcgcctc aaccagccca cccacgtcaa caacggcaac tacacgctgc 1141tggctgccaa ccccttcggc caggcctccg cctccatcat ggctgccttc atggacaacc 1201ctttcgagtt caaccccgag gaccccatcc ctgacactaa cagcacatct ggagacccgg 1261tggagaagaa ggacgaaaca ccttttgggg tctcggtggc tgtgggcctg gccgtctttg 1321cctgcctctt cctttctacg ctgctccttg tgctcaacaa atgtggacgg agaaacaagt 1381ttgggatcaa ccgcccggct gtgctggctc cagaggatgg gctggccatg tccctgcatt 1441tcatgacatt gggtggcagc tccctgtccc ccaccgaggg caaaggctct gggctccaag 1501gccacatcat cgagaaccca caatacttca gtgatgcctg tgttcaccac atcaagcgcc 1561gggacatcgt gctcaagtgg gagctggggg agggcgcctt tgggaaggtc ttccttgctg 1621agtgccacaa cctcctgcct gagcaggaca agatgctggt ggctgtcaag gcactgaagg 1681aggcgtccga gagtgctcgg caggacttcc agcgtgaggc tgagctgctc accatgctgc 1741agcaccagca catcgtgcgc ttcttcggcg tctgcaccga gggccgcccc ctgctcatgg 1801tctttgagta tatgcggcac ggggacctca accgcttcct ccgatcccat ggacctgatg 1861ccaagctgct ggctggtggg gaggatgtgg ctccaggccc cctgggtctg gggcagctgc 1921tggccgtggc tagccaggtc gctgcgggga tggtgtacct ggcgggtctg cattttgtgc 1981accgggacct ggccacacgc aactgtctag tgggccaggg actggtggtc aagattggtg 2041attttggcat gagcagggat atctacagca ccgactatta ccgtgtggga ggccgcacca 2101tgctgcccat tcgctggatg ccgcccgaga gcatcctgta ccgtaagttc accaccgaga 2161gcgacgtgtg gagcttcggc gtggtgctct gggagatctt cacctacggc aagcagccct 2221ggtaccagct ctccaacacg gaggcaatcg actgcatcac gcagggacgt gagttggagc 2281ggccacgtgc ctgcccacca gaggtctacg ccatcatgcg gggctgctgg cagcgggagc 2341cccagcaacg ccacagcatc aaggatgtgc acgcccggct gcaagccctg gcccaggcac 2401ctcctgtcta cctggatgtc ctgggctagg gggccggccc aggggctggg agtggttagc 2461cggaatactg gggcctgccc tcagcatccc ccatagctcc cagcagcccc agggtgatct 2521caaagtatct aattcaccct cagcatgtgg gaagggacag gtgggggctg ggagtagagg 2581atgttcctgc ttctctaggc aaggtcccgt catagcaatt atatttatta tcccttgaaa 2641aaaaaaa

Therapeutic Methods and Agents

The invention features methods of treating a cholangiocarcinoma, e.g., acholangiocarcinoma harboring a fusion described herein. The methodsinclude administering a therapeutic agent, e.g., which antagonizes thefunction of FGFR2 or NTRK1. The therapeutic agent can be a smallmolecule, protein, polypeptide, peptide, nucleic acid, e.g., a siRNA,antisense or micro RNA. Exemplary agents and classes of agents areprovided in Table 2.

TABLE 2 Kinase inhibitors Multi-kinase inhibitors Pan-kinase inhibitorsKinase inhibitors having activity for or selectivity for FGFR2 Kinaseinhibitors having activity for or selectivity for NTRK siRNA, antisenseRNA, or other nucleic acid based inhibitors of FGFR2 or NTRK Antagonistsof FGFR2, e.g., antibodies or small molecules that bind FGFR2Antagonists of NTRK1, e.g., antibodies or small molecules that bind NTRKAZD-2171 BGJ398 AZD-4547 BIBF1120 Brivanib Cediranib Dovitinib ENMD-2076Masitinib JNJ42756493 Lenvatinib LY2874455 Ponatinib Pazopanib R406Regorafenib Other therapeutic agents disclosed herein. PD173074 PD173955Danusertib Dovitinib Dilactic Acid TSU-68 Tyrphostin AG 1296 MK-2461Brivanib Alaninate Lestaurtinib K252a PHA-848125 AZ-23 Oxindole-3 AV369bACTB 1003 Volasertib R1530 Loxo-101 ARRY-470 ARRY-786 RXDX-101 RXDX-102

These treatments can be provided to a patient having had unsatisfactoryresponse to a cytotoxic chemotherapy or opportunistic resection.

An agent from Table 2 can be administered, alone or in combination,e.g., in combination with other chemotherapeutic agents or procedures,in an amount sufficient to reduce or inhibit the tumor cell growth,and/or treat or prevent the cancer(s), in the subject.

Exemplary agents are discussed in more detail below.

Regorafenib

Regorafenib is a multi-kinase inhibitor that inhibits multiplemembrane-bound and intracellular kinases, including those in the RET,VEGFR1/2/3, KIT, PDGFR, FGFR1/2, and RAF pathways. Regorafenib has beenapproved to treat patients with metastatic colorectal cancer who havebeen previously treated with fluoropyrimidine-, oxaliplatin-, andirinotecan-based chemotherapy, an anti-VEGF therapy, and, if KRAS wildtype, an anti-EGFR therapy. Tumors with Fgfr2 activation may besensitive to regorafenib. Regorafenib is being studied in clinicaltrials for multiple solid tumor types.

In some embodiments, the kinase inhibitor is regorafenib. Regorafenib(STIVARGA, Bayer) is a small molecule inhibitor of multiplemembrane-bound and intracellular kinases. In in vitro biochemical orcellular assays, regorafenib or its major human active metabolites M-2and M-5 inhibited the activity of FGFR1 and FGFR-2 as well as multipleother kinases. STIVARGA Product Label dated Sep. 27, 2012. Regorafenibhas the chemical name:1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea;and has the following structure:

Ponatinib

Ponatinib is a multi-kinase inhibitor targeting BCR-ABL, as well asVEGFRs and FGFRs. Ponatinib has been approved by the FDA for use inchronic myeloid leukemia (CML) and Philadelphia chromosome-positiveacute lymphoblastic leukemia (ALL). Activating mutations oramplification of FGFR2 can result in sensitivity to ponatinib (Gozgit etal., 2012, Mol. Cancer Ther., 11(3):690-699).

In some embodiments, the kinase inhibitor is ponatinib (AP24534,ICLUSIG, Ariad). Ponatinib is a small molecule kinase inhibitor.Ponatinib inhibited the in vitro tyrosine kinase activity of ABL andT315I mutant ABL with IC50 concentrations of 0.4 and 2.0 nM,respectively. Ponatinib inhibited the in vitro activity of additionalkinases with IC50 concentrations between 0.1 and 20 nM, includingmembers of the PDGFR, FGFR, EPH receptors and SRC families of kinases,and KIT, RET, TIE2, and FLT3. ICLUSIG Product Label dated Dec. 14, 2012.Ponatinib has the chemical name:3-(2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide;and has the following structure:

AZD-4547

In certain embodiments the kinase inhibitor is AZD-4547. AZD-4547 is anorally bioavailable small molecule inhibitor of the fibroblast growthfactor receptor (FGFR). AZD-4547 binds to and inhibits FGFR1, 2 and 3tyrosine kinases. FGFR, up-regulated in many tumor cell types, is areceptor tyrosine kinase essential to tumor cellular proliferation,differentiation and survival. AZD4547 is under clinical investigationfor the treatment of FGFR-dependent tumors. AZD-4547 has the chemicalnameN-(5-(3,5-dimethoxyphenethyl)-1H-pyrazol-3-yl)-4-((3S,5R)-3,5-dimethylpiperazin-1-yl)benzamide;and has the following structure:

BGJ398

In some embodiments, the kinase inhibitor is BGJ398. BGJ398 (NVP-BGJ398)is a potent, selective, and orally bioavailable small molecule inhibitorof the FGFR2 tyrosine kinases. BGJ398 inhibits the proliferation ofvarious FGFR-dependent cell lines including breast and lung cancersharboring FGFR1 amplification, FGFR2-amplified gastric cancer cell linesand FGFR3-mutated bladder cancers. BGJ398 has the chemical name3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6-(4-(4-ethylpiperazin-1-yl)phenylamino)pyrimidin-4-yl)-1-methylurea;and has the following structure:

Masitinib

In some embodiments, the kinase inhibitor is masitinib. Masitinib(AB1010) (commercial names Masivet, Kinavet) is a small moleculetyrosine-kinase inhibitor that is used in the treatment of mast celltumors in animals, particularly dogs. Masitinib inhibits the receptortyrosine kinase c-Kit, as well as the platelet derived growth factorreceptor (PDGFR) and fibroblast growth factor receptor (FGFR). Masitinibhas the chemical nameN-(4-methyl-3-(4-(pyridin-3-yl)thiazol-2-ylamino)phenyl)-4-((4-methylpiperazin-1-yl)methyl)benzamide;and has the following structure:

Lenvatinib

In some embodiments, the kinase inhibitor is Lenvatinib (E7080).Lenvatinib is a small molecule multi-kinase inhibitor that is beinginvestigated for the treatment of various types of cancer by Eisai Co.It inhibits multiple receptor tyrosine kinases including VEGF, FGF andSCF receptors. Lenvatinib (E7080) has the chemical name:1-(4-(6-carbamoyl-7-methoxyquinolin-4-yloxy)-2-chlorophenyl)-3-cyclopropylurea;and has the following structure:

Dovitinib

In some embodiments, the kinase inhibitor is dovitinib. Dovitinib(dovitinib lactate, also known as receptor tyrosine kinase inhibitorTKI258; code names: TKI258 or CHIR-258) is an orally bioavailablelactate salt of a benzimidazole-quinolinone compound. Dovitinib stronglybinds to fibroblast growth factor receptor 3 (FGFR3) and inhibits itsphosphorylation. In addition, dovitinib may inhibit other members of theRTK superfamily, including the vascular endothelial growth factorreceptor; fibroblast growth factor receptor 1; platelet-derived growthfactor receptor type 3; FMS-like tyrosine kinase 3; stem cell factorreceptor (c-KIT); and colony-stimulating factor receptor 1. See NationalCancer Institute Drug Dictionary atcancer.gov/drugdictionary?cdrid=488976. Dovitinib has the chemical name:1-amino-5-fluoro-3-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)quinolin-2(1H)-one;and has the following structure:

Dovitinib Dilactic Acid

In some embodiments, the kinase inhibitor is dovitinib dilactic acid(TKI258 dilactic acid). Dovitinib dilactic acid is a multitargeted RTKinhibitor, mostly for class III (FLT3/c-Kit) with IC50 of 1 nM/2 nM,also potent to class IV (FGFR1/3) and class V (VEGFR1-4) RTKs with IC50from 8-13 nM, less potent to InsR, EGFR, c-Met, EphA2, Tie2, IGFR1 andHER2. Dovitinib dilactic acid has the chemical name: Propanoic acid,2-hydroxy-, compd. with4-amino-5-fluoro-3-[6-(4-methyl-1-piperazinyl)-1H-benzimidazol-2-yl]-2(1H)-quinolinone;and has the following structure:

Brivanib

In some embodiments, the kinase inhibitor is brivanib (BMS-540215).Brivanib is the alaninate salt of the VEGFR-2 inhibitor BMS-540215 andis hydrolyzed to the active moiety BMS-540215 in vivo. BMS-540215, adual tyrosine kinase inhibitor, shows potent and selective inhibition ofVEGFR and fibroblast growth factor receptor (FGFR) tyrosine kinases.Brivanib has the chemical name:(R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[1,2-][1,2,4]triazin-6-yloxy)propan-2-ol;and has the following structure:

ENMD-2076

In certain embodiments the kinase inhibitor is ENMD-2076. ENMD-2076 isorally bioavailable small molecule inhibitor of the Aurora kinase A, aswell as kinases involved in angiogenesis (VEGFRs, FGFRs). The mechanismof action of ENMD-2076 involves several pathways key to tumor growth andsurvival: angiogenesis, proliferation, and the cell cycle. ENMD-2076 hasreceived orphan drug designation from the United States Food and DrugAdministration (the “FDA”) for the treatment of ovarian cancer, multiplemyeloma and acute myeloid leukemia (“AML”). ENMD-2076 has the chemicalname(E)-N-(5-methyl-1H-pyrazol-3-yl)-6-(4-methylpiperazin-1-yl)-2-styrylpyrimidin-4-amine;and has the following structure:

Cediranib

In some embodiments, the kinase inhibitor is Cediranib. Cediranib (alsoRecentin or AZD2171) is a small molecule inhibitor of vascularendothelial growth factor (VEGF) receptor tyrosine kinases. See, e.g.,WO 2007/060402. Cediranib also inhibits platelet derived growth factor(PDGFR)-associated kinases c-Kit, PDGFR-α, and PDGFR-β. Cediranib alsoinhibits FGFR-1 and FGFR-4. Brave, S. R. Molecular Cancer Ther, 10(5):861-873, published online Mar. 25, 2011, doi: 10.1158/1535-71634.Cediranib has the chemical name4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline;and has the following structure:

BIBF 1120

In some embodiments, the kinase inhibitor is BIBF1120 (Nintedanib). BIBF1120 (Nintedanib) is an indolinone derivative that inhibits the processof blood vessel formation (angiogenesis) in tumors. See, e.g.,WO2001/27081; WO2004/13099; WO2010/081817. It potently blocks the VEGFreceptor (VEGFR), PDGFR and fibroblast growth factor receptor (FGFR)kinase activity in enzymatic assays (IC(50), 20-100 nmol/L). BIBF 1120inhibits mitogen-activated protein kinase and Akt signaling pathways inthree cell types contributing to angiogenesis, endothelial cells,pericytes, and smooth muscle cells, resulting in inhibition of cellproliferation (EC(50), 10-80 nmol/L) and apoptosis. BIBF1120 has thechemical name: (Z)-methyl3-((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenylamino)(phenyl)methylene)-2-oxoindoline-6-carboxylate;and has the following structure:

LY2874455

In some embodiments, the kinase inhibitor is LY2874455. LY2874455 is asmall molecule that inhibits all four FGFRs with a similar potency inbiochemical assays. It exhibits potent activity againstFGF/FGFR-mediated signaling in several cancer cell lines and shows abroad spectrum of antitumor activity in several tumor xenograft modelsrepresenting the major FGF/FGFR2 relevant tumor histologies includinglung, gastric, and bladder cancers and multiple myeloma. LY2874455exhibits a 6- to 9-fold in vitro and in vivo selectivity on inhibitionof FGF-over VEGF-mediated target signaling in mice. Furthermore,LY2874455 did not show VEGF receptor 2-mediated toxicities such ashypertension at efficacious doses. See Zhao, G. et al. Mol Cancer Ther.2011 November; 10(11):2200-10. doi: 10.1158/1535-7163. LY2874455 has thechemical name(R)-(E)-2-(4-(2-(5-(1-(3,5-Dichloropyridin-4-yl)ethoxy)-1H-indazol-3yl)vinyl)-1H-pyrazol-1-yl)ethanol;and has the following structure:

JNJ42756493

In some embodiments, the kinase inhibitor is JNJ42756493. JNJ42756493 isan orally bioavailable, pan fibroblast growth factor receptor (FGFR)inhibitor. Upon oral administration, JNJ-42756493 binds to and inhibitsFGFR, which may result in the inhibition of FGFR-related signaltransduction pathways and thus the inhibition of tumor cellproliferation and tumor cell death in FGFR-overexpressing tumor cells.

Pazopanib

In some embodiments, the kinase inhibitor is pazopanib. Pazopanib(Votrient) is a potent and selective multi-targeted receptor tyrosinekinase inhibitor. The FDA has approved it for renal cell carcinoma andsoft tissue sarcoma. Pazopanib has the chemical name:5-[[4-[(2,3-dimethyl-2H-indazol-6yl)methylamino]-2-pyrimidinyl]amino]-2-methylbenzenesulfonamidemonohydrochloride; and has the following structure:

PD-173955

In some embodiments, the kinase inhibitor is PD-173955. PD-173955 is apotent tyrosine kinase inhibitor. PD-173955 is a src tyrosine kinaseinhibitor. PD173955 inhibited Bcr-Abl-dependent cell growth. PD173955showed cell cycle arrest in G(1). PD173955 has an IC(50) of 1-2 nM inkinase inhibition assays of Bcr-Abl, and in cellular growth assays itinhibits Bcr-Abl-dependent substrate tyrosine phosphorylation. PD173955inhibited kit ligand-dependent c-kit autophosphorylation(IC(50)=approximately 25 nM) and kit ligand-dependent proliferation ofM07e cells (IC(50)=40 nM) but had a lesser effect on interleukin3-dependent (IC(50)=250 nM) or granulocyte macrophage colony-stimulatingfactor (IC(50)=1 microM)-dependent cell growth. PD-173955 has thechemical name:6-(2,6-dichlorophenyl)-8-methyl-2-(3-methylsulfanylanilino)pyrido[2,3-d]pyrimidin-7-one;and has the following structure:

R406

In some embodiments, the kinase inhibitor is R406. R406 is a potenttyrosine kinase inhibitor. R406 is a potent Syk inhibitor with IC50 of41 nM, strongly inhibits Syk but not Lyn, 5-fold less potent to Flt3.R406 has the chemical name:6-(2,6-dichlorophenyl)-8-methyl-2-(3-methylsulfanylanilino)pyrido[2,3-d]pyrimidin-7-one;and has the following structure:

PD173074

In some embodiments, the kinase inhibitor is PD173074. PD173074 is apotent FGFR1 inhibitor with IC50 of −25 nM and also inhibits VEGFR2 withIC50 of 100-200 nM, −1000-fold selective for FGFR1 than PDGFR and c-Src.PD173074 has the chemical name:1-tert-butyl-3-(2-(4-(diethylamino)butylamino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl)urea;and has the following structure:

Danusertib

In some embodiments, the kinase inhibitor is danusertib (PHA-739358).Danusertib is an Aurora kinase inhibitor for Aurora A/B/C with IC50 of13 nM/79 nM/61 nM, modestly potent to Abl, TrkA, c-RET and FGFR1, andless potent to Lck, VEGFR2/3, c-Kit, and CDK2. Danusertib has thechemical name:(R)—N-(5-(2-methoxy-2-phenylacetyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-4-(4-methylpiperazin-1-yl)benzamide;and has the following structure:

TSU-680

In some embodiments, the kinase inhibitor is TSU-68 (SU6668). SU6668 hasgreatest potency against PDGFR autophosphorylation with K, of 8 nM, butalso strongly inhibits Flk-1 and FGFR1 trans-phosphorylation, littleactivity against IGF-1R, Met, Src, Lck, Zap70, Abl and CDK2; and doesnot inhibit EGFR. SU6668 has the chemical name:(Z)-3-(2,4-dimethyl-5-((2-oxoindolin-3-ylidene)methyl)-1H-pyrrol-3-yl)propanoicacid; and has the following structure:

Tyrphostin AG 1296 iIn some embodiments, the kinase inhibitor istyrphostin AG 1296 (AG 1296). Tyrphostin AG 1296 (AG 1296) is aninhibitor of PDGFR with IC50 of 0.3-0.5 μM, no activity to EGFR.Tyrphostin AG 1296 has the chemical name Quinoxaline,6,7-dimethoxy-2-phenyl-; and has the following structure:

MK-2461

In some embodiments, the kinase inhibitor is MK-2461. MK-2461 is apotent, multi-targeted inhibitor for c-Met(WT/mutants) with IC50 of0.4-2.5 nM, less potent to Ron, Flt1; 8- to 30-fold greater selectivityof c-Met targets versus FGFR1, FGFR2, FGFR3, PDGFRβ, KDR, Flt3, Flt4,TrkA, and TrkB. MK-2461 has the chemical name:N-((2R)-1,4-Dioxan-2-ylmethyl)-N-methyl-N′-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]sulfamide;and has the following structure:

Brivanib Alaninate

In some embodiments, the kinase inhibitor is brivanib alaninate(BMS-582664). Brivanib alaninate (BMS-582664) is the prodrug ofBMS-540215, an ATP-competitive inhibitor against VEGFR2 with IC50 of 25nM. Brivanib alaninate has the chemical name:(S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[1,2-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate; and has the following structure:

Lestaurtinib

In certain embodiments the kinase inhibitor is lestaurtinib.Lestaurtinib is a potent JAK2, FLT3 and TrkA inhibitor (IC₅₀ values are0.9, 3 and <25 nM respectively) that prevents STATS phosphorylation(IC₅₀=20-30 nM). Exhibits antiproliferative activity in vitro(IC₅₀=30-100 nM in HEL92.1.7 cells) and is effective againstmyeloproliferative disorders in vivo. Lestaurtinib has the chemical name(9S,10S,12R)-2,3,9,10,11,12-Hexahydro-10-hydroxy-10-(hydroxymethyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one;and has the following structure:

PHA-848125

In certain embodiments the kinase inhibitor is PHA-848125 (Milciclib).Milciclib is an orally bioavailable inhibitor of cyclin-dependentkinases (CDKs) and thropomyosin receptor kinase A (TRKA), with potentialantineoplastic activity. CDK2/TRKA inhibitor PHA-848125 AC potentlyinhibits cyclin-dependent kinase 2 (CDK2) and exhibits activity againstother CDKs including CDK1 and CDK4, in addition to TRKA. PHA-848125(Milciclib) has the chemical name:N,1,4,4-tetramethyl-8-((4-(4-methylpiperazin-1-yl)phenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide;and has the following structure:

K252a

In certain embodiments the kinase inhibitor is K252a. K252a is an analogof Staurosporine (Cat. No. 1048) that acts as a non-selective proteinkinase inhibitor. Inhibits PKA (Ki=18 nM), PKC (Ki=25 nM), and PKG(Ki=20 nM). Potently inhibits CaMK (Ki=1.8 nM), competitively with ATPand noncompetitively with the substrate. K252a has the followingstructure:

AZ-23

In certain embodiments the kinase inhibitor is AZ-23. AZ-23 is a potentand selective tyrosine kinase Trk inhibitor with IC50 to 2 and 8 nM forTrkA and TrkB respectively; AZ-23 showed in vivo TrkA kinase inhibitionand efficacy in mice following oral administration; having potential fortherapeutic utility in neuroblastoma and multiple other cancerindications. AZ-23 has the chemical name5-chloro-N-[(1S)-1-(5-fluoropyridin-2-yl)ethyl]-N′-(5-propan-2-yloxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine;and has the following structure:

Oxindole 3

In certain embodiments the kinase inhibitor is oxindole 3. Oxindole 3has the chemical name: 1,2 Dihydro-3H-indol-3-one; and has the followingstructure:

In other embodiments, the inhibitor is a pan FGFR inhibitor. Forexample, the inhibitor is ACTB-1003 as described in Burd, A. et al.(2010) EJC Supplements Vol. 8(7): page 51; Patel, K. et al. (2010)Journal of Clinical Oncology, ASCO Annual Meeting Abstracts. Vol 28, No15_suppl (May 20 Supplement), 2010: e13665.

In other embodiments, the inhibitor is an oral inhibitor.

In other embodiments, the inhibitor is Volasertib. Volasertib has thefollowing chemical structure:

In another embodiment, the inhibitor is R1530. R1530 is apyrazolobenzodiazepine small molecule with potential antiangiogenesisand antineoplastic activities. R1530 is also a mitosis-angiogenesisinhibitor (MAI) that inhibits multiple receptor tyrosine kinasesinvolved in angiogenesis, such as vascular endothelial growth factorreceptor (VEGFR)-1, -2, -3, platelet-derived growth factor receptor(PDGFR) beta, FMS-like tyrosine kinase (Flt)-3, and fibroblast growthfactor receptor (FGFR)-1, -2. In addition, this agents exhibitsanti-proliferative activity by initiating mitotic arrest and inducingapoptosis. R1530 has a chemical name:5-(2-chlorophenyl)-7-fluoro-8-methoxy-3-methyl-2,10-dihydrobenzo[e]pyrazolo[4,3-b][1,4]diazepine(described in, e.g., Kolinsky K, et al. Cancer Chemother Pharmacol. 2011December; 68(6):1585-94. Epub 2011 May 8. PubMed PMID: 21553286).

In another embodiment, the inhibitor is ARRY-470. ARRY-470 has thefollowing structure and chemical name

In another embodiment, the inhibitor is RXDX-101 or RXDX-102. RXDX-101is an orally available, selective tyrosine kinase inhibitor of the TrkA,ROS1 and ALK proteins. RXDX-101 is designed as a targeted therapeuticcandidate to treat patients with cancers that harbor activatingalterations to TrkA, ROS1 and ALK. RXDX-102 is an orally available,selective pan-TRK tyrosine kinase inhibitor, or inhibitor of the TrkA,TrkB and TrkC proteins. RXDX-102 is designed as an oncogene-targetedtherapeutic candidate to treat patients with cancers that harboractivating alterations to TrkA, TrkB or TrkC.

In one embodiment, the therapeutic agent is a kinase inhibitor. Forexample, the kinase inhibitor is a multi-kinase inhibitor or a specificinhibitor. Exemplary kinase inhibitors include, but are not limited to,axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™,AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®),gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib(TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272),nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,SU11248), toceranib (PALLADIA®), vandetanib, vatalanib (PTK787, PTK/ZK),sorafenib (NEXAVAR®), ENMD-2076, PCI-32765, AC220, dovitinib lactate(TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903,PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120(VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154,CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, andXL228.

In other embodiments, the anti-cancer agent inhibits the expression ofnucleic acid encoding fusions. Examples of such antagonists includenucleic acid molecules, for example, antisense molecules, ribozymes,RNAi, triple helix molecules that hybridize to a nucleic acid encoding afusion, and blocks or reduces mRNA expression of a fusion.

In other embodiments, the kinase inhibitor is administered incombination with a second therapeutic agent or a different therapeuticmodality, e.g., anti-cancer agents, and/or in combination with surgicaland/or radiation procedures.

In yet another embodiment, the inhibitor is an antibody molecule (e.g.,an antibody or an antigen-binding fragment thereof). In one embodiment,the antibody molecule binds to FGFR2, e.g., binds to the extracellularligand binding domain of FGFR2. In one embodiment, the antibody moleculebinds to an isoform of FGFR2, e.g., binds to a IIIb-isoform of FGFR2. Inone embodiment, the antibody molecule is AV369b described in Bai et al.(2010) 22^(nd) EORTC-NCI-AACR Symposium, Berlin, Germany 16-19, 2010. Inone embodiment, the antibody molecule: competes for binding, binds to asimilar epitope as AV369b and/or has one or more of the properties asAV369b.

By “in combination with,” it is not intended to imply that the therapyor the therapeutic agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope of the invention. The pharmaceutical compositions canbe administered concurrently with, prior to, or subsequent to, one ormore other additional therapies or therapeutic agents. In general, eachagent will be administered at a dose and/or on a time scheduledetermined for that agent. In will further be appreciated that theadditional therapeutic agent utilized in this combination can beadministered together in a single composition or administered separatelyin different compositions. The particular combination to employ in aregimen will take into account compatibility of the inventivepharmaceutical composition with the additional therapeutically activeagent and/or the desired therapeutic effect to be achieved.

“Treat,” “treatment,” and other forms of this word refer to theadministration of a kinase inhibitor, alone or in combination with asecond agent to impede growth of a cancer, to cause a cancer to shrinkby weight or volume, to extend the expected survival time of the subjectand or time to progression of the tumor or the like. In those subjects,treatment can include, but is not limited to, inhibiting tumor growth,reducing tumor mass, reducing size or number of metastatic lesions,inhibiting the development of new metastatic lesions, prolongedsurvival, prolonged progression-free survival, prolonged time toprogression, and/or enhanced quality of life.

As used herein, unless otherwise specified, the terms “prevent,”“preventing” and “prevention” contemplate an action that occurs before asubject begins to suffer from the re-growth of the cancer and/or whichinhibits or reduces the severity of the cancer.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment or management of the cancer, or todelay or minimize one or more symptoms associated with the cancer. Atherapeutically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other therapeuticagents, which provides a therapeutic benefit in the treatment ormanagement of the cancer. The term “therapeutically effective amount”can encompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of the cancer, or enhances the therapeutic efficacyof another therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to preventre-growth of the cancer, or one or more symptoms associated with thecancer, or prevent its recurrence. A prophylactically effective amountof a compound means an amount of the compound, alone or in combinationwith other therapeutic agents, which provides a prophylactic benefit inthe prevention of the cancer. The term “prophylactically effectiveamount” can encompass an amount that improves overall prophylaxis orenhances the prophylactic efficacy of another prophylactic agent.

As used herein, the term “patient” or “subject” refers to an animal,typically a human (i.e., a male or female of any age group, e.g., apediatric patient (e.g, infant, child, adolescent) or adult patient(e.g., young adult, middle-aged adult or senior adult) or other mammal,such as a primate (e.g., cynomolgus monkey, rhesus monkey). When theterm is used in conjunction with administration of a compound or drug,then the patient has been the object of treatment, observation, and/oradministration of the compound or drug.

Isolated Nucleic Acid Molecules

One aspect featured in the invention pertains to isolated nucleic acidmolecules that include a fusion, including nucleic acids which encode afusion polypeptide or a portion of such a polypeptide. The nucleic acidmolecules include those nucleic acid molecules which reside in genomicregions identified herein. As used herein, the term “nucleic acidmolecule” includes DNA molecules (e.g., cDNA or genomic DNA) and RNAmolecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded; in certain embodiments the nucleic acid molecule isdouble-stranded DNA.

Isolated nucleic acid molecules also include nucleic acid moleculessufficient for use as hybridization probes or primers to identifynucleic acid molecules that correspond to a fusion, e.g., those suitablefor use as PCR primers for the amplification or mutation of nucleic acidmolecules.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. In certain embodiments, an “isolated” nucleicacid molecule is free of sequences (such as protein-encoding sequences)which naturally flank the nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organismfrom which the nucleic acid is derived. For example, in variousembodiments, the isolated nucleic acid molecule can contain less thanabout 5 kB, less than about 4 kB, less than about 3 kB, less than about2 kB, less than about 1 kB, less than about 0.5 kB or less than about0.1 kB of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

The language “substantially free of other cellular material or culturemedium” includes preparations of nucleic acid molecule in which themolecule is separated from cellular components of the cells from whichit is isolated or recombinantly produced. Thus, nucleic acid moleculethat is substantially free of cellular material includes preparations ofnucleic acid molecule having less than about 30%, less than about 20%,less than about 10%, or less than about 5% (by dry weight) of othercellular material or culture medium.

A fusion nucleic acid molecule can be isolated using standard molecularbiology techniques and the sequence information in the database recordsdescribed herein. Using all or a portion of such nucleic acid sequences,fusion nucleic acid molecules as described herein can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y, 1989).

A fusion nucleic acid molecule can be amplified using cDNA, mRNA, orgenomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acidmolecules so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule featured inthe invention can be prepared by standard synthetic techniques, e.g.,using an automated DNA synthesizer.

In another embodiment, a fusion nucleic acid molecule comprises anucleic acid molecule which has a nucleotide sequence complementary tothe nucleotide sequence of the fusion nucleic acid molecule or to thenucleotide sequence of a nucleic acid encoding a fusion protein. Anucleic acid molecule which is complementary to a given nucleotidesequence is one which is sufficiently complementary to the givennucleotide sequence that it can hybridize to the given nucleotidesequence thereby forming a stable duplex.

Moreover, a fusion nucleic acid molecule can comprise only a portion ofa nucleic acid sequence, wherein the full length nucleic acid sequenceor which encodes a fusion polypeptide. Such nucleic acid molecules canbe used, for example, as a probe or primer. The probe/primer typicallyis used as one or more substantially purified oligonucleotides. Theoligonucleotide typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 7, at leastabout 15, at least about 25, at least about 50, at least about 75, atleast about 100, at least about 125, at least about 150, at least about175, at least about 200, at least about 250, at least about 300, atleast about 350, at least about 400, at least about 500, at least about600, at least about 700, at least about 800, at least about 900, atleast about 1 kb, at least about 2 kb, at least about 3 kb, at leastabout 4 kb, at least about 5 kb, at least about 6 kb, at least about 7kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, atleast about 15 kb, at least about 20 kb, at least about 25 kb, at leastabout 30 kb, at least about 35 kb, at least about 40 kb, at least about45 kb, at least about 50 kb, at least about 60 kb, at least about 70 kb,at least about 80 kb, at least about 90 kb, at least about 100 kb, atleast about 200 kb, at least about 300 kb, at least about 400 kb, atleast about 500 kb, at least about 600 kb, at least about 700 kb, atleast about 800 kb, at least about 900 kb, at least about 1 mb, at leastabout 2 mb, at least about 3 mb, at least about 4 mb, at least about 5mb, at least about 6 mb, at least about 7 mb, at least about 8 mb, atleast about 9 mb, at least about 10 mb or more consecutive nucleotidesof a fusion nucleic acid.

In another embodiment, an isolated fusion nucleic acid molecule is atleast 7, at least 15, at least 20, at least 25, at least 30, at least35, at least 40, at least 45, at least 50, at least 55, at least 60, atleast 65, at least 70, at least 75, at least 80, at least 85, at least90, at least 95, at least 100, at least 125, at least 150, at least 175,at least 200, at least 250, at least 300, at least 350, at least 400, atleast 450, at least 550, at least 650, at least 700, at least 800, atleast 900, at least 1000, at least 1200, at least 1400, at least 1600,at least 1800, at least 2000, at least 2200, at least 2400, at least2600, at least 2800, at least 3000, or more nucleotides in length andhybridizes under stringent conditions to a fusion nucleic acid moleculeor to a nucleic acid molecule encoding a protein corresponding to amarker featured in the invention.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, or at least 85% identical to each othertypically remain hybridized to each other. Such stringent conditions areknown to those skilled in the art and can be found in sections6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989). Another, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C.

The invention also includes molecular beacon nucleic acid moleculeshaving at least one region which is complementary to a fusion nucleicacid molecule, such that the molecular beacon is useful for quantitatingthe presence of the nucleic acid molecule featured in the invention in asample. A “molecular beacon” nucleic acid is a nucleic acid moleculecomprising a pair of complementary regions and having a fluorophore anda fluorescent quencher associated therewith. The fluorophore andquencher are associated with different portions of the nucleic acid insuch an orientation that when the complementary regions are annealedwith one another, fluorescence of the fluorophore is quenched by thequencher. When the complementary regions of the nucleic acid moleculesare not annealed with one another, fluorescence of the fluorophore isquenched to a lesser degree. Molecular beacon nucleic acid molecules aredescribed, for example, in U.S. Pat. No. 5,876,930.

In one embodiment, a fusion includes an in-frame fusion of an exon offibroblast growth factor receptor 2 (FGFR2), e.g., one more exons ofFGFR2 (e.g., one or more of exons 1-16 of FGFR2) or a fragment thereof,and an exon of a partner as set forth in FIG. 1B (e.g., a transforming,acidic coiled-coil containing protein 3 (TACC3), e.g., one or more exonsof a TACC3 (e.g., one or more of exons 11-16 of TACC3) or a fragmentthereof. In other embodiments, one or more exons of KIAA1598, BICC1,PARK2, NOL4 or ZDHHC6 are fused as summarized in FIGS. 1A-1C. Forexample, the FGFR3-TACC3 fusion can include an in-frame fusion within anintron of FGFR2 or a fragment thereof, with an intron of TACC3,KIAA1598, BICC1 PARK2, NOL4 or ZDHHC6, or a fragment thereof, asdepicted in FIG. 1A. In one embodiment, the fusion of the FGFR2-fusioncomprises the nucleotide sequence of: chromosome 10 at one or more ofthe nucleotides shown in FIG. 1A (plus or minus 10, 20, 30, 50, 60, 70,80, 100 or more nucleotides) and a partner in chromosome 4 or 10 at oneor more of the nucleotides shown in FIG. 1A (plus or minus 10, 20, 30,50, 60, 70, 80, 100 or more nucleotides). In one embodiment, theFGFR3-TACC3 fusion is a translocation, e.g., a translocation of aportion of chromosome 10 and 4. In another embodiment, theFGFR2-KIAA1598 fusion is a deletion, e.g., a deletion of chromosome 10.In another embodiment, the FGFR2-BICC1 fusion is an inversion, e.g., aan inversion of chromosome 10 (e.g., as summarized in FIG. 1A).

In certain embodiments, the FGFR2-TACC3, FGFR2-KIAA1598, FGFR2-BICC1,BICC1-FGFR2, PARK2-FGFR2, FGFR2-NOL4, or ZDHHC6-FGFR2 fusion is in a 5′-to 3′-configuration (also referred to herein as, for example,“5′-FGFR2-TACC-3′).” The term “fusion” or “fusion molecule” can refer toa polypeptide or a nucleic acid fusion, depending on the context. It mayinclude a full-length sequence or a fragment thereof, e.g., a fusionjunction (e.g., a fragment including a portion of FGFR2 and a portion ofTAC3, KIAA1598, BICC1 PARK2, NOL4 or ZDHHC6, e.g., a portion of theFGFR3-TACC3, FGFR2-KIAA1598, FGFR2-BICC1, BICC1-FGFR2 PARK2-FGFR2,FGFR2-NOL4, or ZDHHC6-FGFR2 fusion described herein). In one embodiment,the FGFR2-TACC3 fusion polypeptide includes the amino acid sequenceshown in FIG. 3 (SEQ ID NO:2) and/or FIG. 5 (SEQ ID NO:4), or an aminoacid sequence substantially identical thereto. In another embodiment,the FGFR2-TACC3 fusion nucleic acid includes the nucleotide sequenceshown in FIGS. 2A-2B (SEQ ID NO:1) and/or FIGS. 4A-4B (SEQ ID NO:3), ora nucleotide sequence substantially identical thereto. In anotherembodiment, the FGFR2-KIAA1598 fusion polypeptide includes the aminoacid sequence shown in FIG. 3 (SEQ ID NO:2) and/or FIG. 7 (SEQ ID NO:6),or an amino acid sequence substantially identical thereto. In anotherembodiment, the FGFR2-KIAA1598 fusion nucleic acid includes thenucleotide sequence shown in FIGS. 2A-2B (SEQ ID NO:1) and/or FIGS.6A-6C (SEQ ID NO:5), or a nucleotide sequence substantially identicalthereto. In another embodiment, the FGFR2-BICC1 fusion polypeptideincludes the amino acid sequence shown in FIG. 3 (SEQ ID NO:2) and/orFIG. 9 (SEQ ID NO:8), or an amino acid sequence substantially identicalthereto. In another embodiment, the FGFR2-BICC1 fusion nucleic acidincludes the nucleotide sequence shown in FIG. 2 (SEQ ID NO:1) and/orFIG. 8 (SEQ ID NO:7), or a nucleotide sequence substantially identicalthereto. In another embodiment, the BICC1-FGFR2 fusion polypeptideincludes the amino acid sequence shown in FIG. 9 (SEQ ID NO:8) and/orFIG. 3 (SEQ ID NO:2), or an amino acid sequence substantially identicalthereto. In another embodiment, the BICC1-FGFR2 fusion nucleic acidincludes the nucleotide sequence shown in FIGS. 8A-8B (SEQ ID NO:7)and/or FIGS. 2A-2B (SEQ ID NO:1), or a nucleotide sequence substantiallyidentical thereto. In another embodiment, the PARK2-FGFR2 fusionpolypeptide includes the amino acid sequence shown in FIG. 11 (SEQ IDNO:10) and/or FIG. 13 (SEQ ID NO:12), or an amino acid sequencesubstantially identical thereto. In another embodiment, the FGFR2-NOL4fusion polypeptide includes the amino acid sequence shown in FIG. 15(SEQ ID NO:14) and/or FIG. 13 (SEQ ID NO:12), or an amino acid sequencesubstantially identical thereto. In another embodiment, the ZDHHC6-FGFR2fusion polypeptide includes the amino acid sequence shown in FIG. 16(SEQ ID NO:15) and/or FIG. 13 (SEQ ID NO:12), or an amino acid sequencesubstantially identical thereto.

In one embodiment, the FGFR2 fusion polypeptide comprises sufficientFGFR2 and sufficient partner sequence such that the fusion has kinaseactivity, e.g., has elevated activity, e.g., FGFR2 tyrosine kinaseactivity, as compared with wild type FGFR2, e.g., in a cell of a cancerreferred to herein (a cholangiocarcinoma). In one embodiment, thepartner, e.g., TACC3 sequence, has a coiled-coil domain, e.g., it maydimerize with one or more partners.

In certain embodiments, the FGFR2-TACC3 fusion comprises one or more (orall of) exons 1-16 from FGFR2 and one or more (or all of) exons 11-16from TACC3 (e.g., one or more of the exons shown in FIGS. 2-5 ). Incertain embodiments, the FGFR2-TACC3 fusion comprises at least 1, 2, 3,4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16 or more exons from FGFR2 andat least 1, 2, 3, 4, 5, 6, 7, 8, or more exons from TACC3 (e.g., fromthe FGFR3 and TACC3 sequences shown in FIGS. 2-5 (SEQ ID NOs:1-4).

In certain embodiments, the FGFR2-KIAA1598 fusion comprises one or more(or all of) exons 1-16 from FGFR2 and one or more (or all of) exons 7-17from KIAA1598 (e.g., one or more of the exons shown in FIGS. 2-3 and 6-7). In certain embodiments, the FGFR2-KIAA1598 fusion comprises at least1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16 or more exons fromFGFR2 and at least 1, 2, 3, 4, 5, 6, 7, 8, or more exons from KIAA1598(e.g., from the FGFR2 and KIAA1598 sequences shown in FIGS. 2-3 and 6-7(SEQ ID NOs:1-2 and 5-6).

In certain embodiments, the FGFR2-BICC1 fusion comprises one or more (orall of) exons 1-16 from FGFR2 and one or more (or all of) exons 18-21from BICC1 (e.g., one or more of the exons shown in FIGS. 2-3 and 8-9 ).In certain embodiments, the FGFR2-BICC1 fusion comprises at least 1, 2,3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16 or more exons from FGFR2and at least 1, 2, 3, 4, or more exons from BICC1 (e.g., from the FGFR2and BICC1 sequences shown in FIGS. 2-3 and 8-9 (SEQ ID NOs:1-2 and 7-8).

In certain embodiments, the BICC1-FGFR2-fusion comprises one or more (orall of) exons 1-2 FROM BICC1 and exon 17 from FGFR2 (e.g., one or moreof the exons shown in FIGS. 2-3 and 8-9 ) (e.g., from the FGFR2 andBICC1 sequences shown in FIGS. 2-3 and 8-9 (SEQ ID NOs:1-2 and 7-8).

In certain embodiments, the PARK2-FGFR2-fusion comprises one or more (orall of) exons 1-9 of PARK2 and exon 18 from FGFR2 (e.g., one or more ofthe exons shown in FIGS. 10-11 and 12-13 ) (e.g., from the PARK2 andFGFR2 sequences shown in FIGS. 10-11 and 12-13 (SEQ ID NOs:9-10 and11-12).

In certain embodiments, the FGFR2-NOL4-fusion comprises one or more (orall of) exons 1-17 of FGFR2 and exon 7-11 from NOL4 (e.g., one or moreof the exons shown in FIGS. 12-13 and 14-15 and) (e.g., from the FGFR2and NOL4 sequences shown in FIGS. 12-13 and 14-15 (SEQ ID NOs: 11-12 and13-14).

In certain embodiments, the ZDHHC6-FGFR2-fusion comprises one or more(or all of) exons 1-5 of ZDHHC6 and exon 18 from FGFR2 (e.g., one ormore of the exons shown in FIGS. 16-17 and 12-13 ) (e.g., from theZDHHC6 and FGFR2 sequences shown in FIGS. 16-17 and 12-13 (SEQ IDNOs:15-16 and 11-12).

FGFR2 Fusion Nucleic Acid Molecules

In one aspect, the invention features a nucleic acid molecule (e.g., anisolated or purified) nucleic acid molecule that includes a fragment ofan FGFR2 gene, and a fragment of a TACC3, KIAA1598, BICC1, PARK2, NOL4or ZDHHC6 as summarized in FIGS. 1A-1C gene. In one embodiment, thenucleotide sequence encodes a FGFR2 fusion polypeptide that includes anFGFR2 tyrosine kinase domain or a functional fragment thereof. Inanother embodiment, the nucleotide sequence encodes a fragment of theFGFR2 polypeptide of SEQ ID NO:2 or 12, or a fragment thereof; or asequence substantially identical thereto. In other embodiments, thenucleic acid molecule includes a fragment of the TACC3 gene encoding theamino acid sequence of SEQ ID NO:4, or a fragment thereof; or a sequencesubstantially identical thereto. In other embodiments, the nucleic acidmolecule includes a fragment of the KIAA1598 gene encoding the aminoacid sequence of SEQ ID NO:6, or a fragment thereof; or a sequencesubstantially identical thereto. In other embodiments, the nucleic acidmolecule includes a fragment of the BICC1 gene encoding the amino acidsequence of SEQ ID NO: 8, or a fragment thereof; or a sequencesubstantially identical thereto. In other embodiments, the nucleic acidmolecule includes a fragment of the PARK2 gene encoding the amino acidsequence of SEQ ID NO:10, or a fragment thereof; or a sequencesubstantially identical thereto. In other embodiments, the nucleic acidmolecule includes a fragment of the NOL4 gene encoding the amino acidsequence of SEQ ID NO:14, or a fragment thereof; or a sequencesubstantially identical thereto. In other embodiments, the nucleic acidmolecule includes a fragment of the ZDHHC6 gene encoding the amino acidsequence of SEQ ID NO:16, or a fragment thereof; or a sequencesubstantially identical thereto.

In one embodiment, the nucleic acid molecule includes a fusion, e.g., anin-frame fusion, between an intron of FGFR2, or a fragment thereof), andan intron of TACC3, KIAA1598, BICC1 PARK2, NOL4 or ZDHHC6, or a fragmentthereof, as depicted in FIG. 1A. The FGFR2 fusion can comprise a fusionof the nucleotide sequence of: chromosome 10 at one or more of thenucleotides depicted in FIG. 1A (plus or minus 10, 20, 30, 50, 60, 70,80, 100 nucleotides) and chromosome 4 or 10 at one or more of thenucleotides depicted in FIG. 1A (plus or minus 10, 20, 30, 50, 60, 70,80, 100 nucleotides), or a fragment thereof. In another embodiment, theFGFR2 fusion comprises a nucleotide sequence shown in FIGS. 2A-2B (SEQID NO: 1) or FIGS. 12A-12B (SEQ ID NO:11) and a partner chosen from:FIGS. 4A-4B (SEQ ID NO:3), or a fragment thereof; FIGS. 6A-6C (SEQ IDNO:5), or a fragment thereof; FIGS. 8A-8B (SEQ ID NO:3), or a fragmentthereof; FIGS. 10A-10B (SEQ ID NO:9), or a fragment thereof; FIGS.14A-14B (SEQ ID NO:13), or a fragment thereof; or FIG. 16 (SEQ IDNO:15), or a fragment thereof.

In one embodiment, the FGFR2 fusion comprises a nucleotide sequencesubstantially identical to the nucleotide sequence shown in FIGS. 2A-2B(SEQ ID NO: 1), or a fragment thereof or FIGS. 12A-12B (SEQ ID NO:11)and a partner chosen from: FIGS. 4A-4B (SEQ ID NO:3), or a fragmentthereof; FIGS. 6A-6C (SEQ ID NO:5), or a fragment thereof; FIGS. 8A-8B(SEQ ID NO:3), or a fragment thereof; FIGS. 10A-10B (SEQ ID NO:9), or afragment thereof; FIGS. 14A-14B (SEQ ID NO:13), or a fragment thereof;or FIG. 16 (SEQ ID NO:15), or a fragment thereof; or a nucleotidesequence at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 99%, at least 99.5 or greater,identical thereto. In one embodiment, the FGFR2 fusion comprises anucleotide sequence containing at least 50, 100, 150, 200, 500, 1000,1500, 2000, 2500, 3000, or more nucleotides of the nucleotide sequenceshown in FIGS. 2A-2B (SEQ ID NO: 1) or FIGS. 12A-12B (SEQ ID NO:11) anda partner chosen from: FIGS. 4A-4B (SEQ ID NO:3), or a fragment thereof;FIGS. 6A-6C (SEQ ID NO:5), or a fragment thereof; FIGS. 8A-8B (SEQ IDNO:3), or a fragment thereof; FIGS. 10A-10B (SEQ ID NO:9), or a fragmentthereof; FIGS. 14A-14B (SEQ ID NO:13), or a fragment thereof; or FIG. 16(SEQ ID NO:15), or a fragment thereof, or a sequence substantiallyidentical thereto.

In certain embodiments, the BICC1-FGFR2-fusion comprises one or more (orall of) exons 1-2 and exon 17 from FGFR2 (e.g., one or more of the exonsshown in FIGS. 2-3 and 8-9 , or a sequence substantially identicalthereto) (e.g., from the FGFR2 and BICC1 sequences shown in FIGS. 2-3and 8-9 (SEQ ID NOs:1-2 and 7-8, or a sequence substantially identicalthereto).

In one embodiment, the nucleic acid molecule is complementary to atleast a portion of a nucleotide sequence disclosed herein, e.g., iscapable of hybridizing under a stringency condition described herein toSEQ ID NO:1 or SEQ ID NO:11 and/or SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:15, or a fragment thereof.In yet another embodiment, the nucleic acid molecule hybridizes to anucleotide sequence that is complementary to at least a portion of anucleotide sequence disclosed herein, e.g., is capable of hybridizingunder a stringency condition to a nucleotide sequence complementary toSEQ ID NO:1 or SEQ ID NO:11 and/or SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:15, or a fragment thereof.

In an embodiment, the FGFR2-TACC3 nucleic acid molecule comprisessufficient FGFR2 and sufficient TACC3 sequence such that the encoded 5′FGFR3-2′ TACC3 fusion has kinase activity, e.g., has elevated activity,e.g., FGFR2 kinase activity, as compared with wild type FGFR2, e.g., ina cell of a cancer referred to herein. In certain embodiments, the 5′FGFR2-3′ TACC3 fusion comprises exons 1-16 from FGFR2 and exon 11-16from TACC3. In certain embodiments, the FGFR3-TACC3 fusion comprises atleast 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, or more exonsfrom FGFR2 and at least 1, 2, 3, 4, 5, 6, 7, 9, 10, or more exons fromTACC3. In certain embodiments, the FGFR2-KIAA1598 fusion comprises oneor more (or all of) exons 1-16 from FGFR2 and one or more (or all of)exons 7-17 from KIAA1598 (e.g., one or more of the exons shown in FIGS.2-3 and 6-7 or a sequence substantially identical thereto). In certainembodiments, the FGFR2-KIAA1598 fusion comprises at least 1, 2, 3, 4, 5,6, 7, 9, 10, 11, 12, 13, 14, 15, 16 or more exons from FGFR2 and atleast 1, 2, 3, 4, 5, 6, 7, 8, or more exons from KIAA1598 (e.g., fromthe FGFR2 and KIAA1598 sequences shown in FIGS. 2-3 and 6-7 (SEQ IDNOs:1-2 and 5-6), or a sequence substantially identical thereto. Incertain embodiments, the FGFR2-BICC1 fusion comprises one or more (orall of) exons 1-16 from FGFR2 and one or more (or all of) exons 18-21from BICC1 (e.g., one or more of the exons shown in FIGS. 2-3 and 8-9 ,or a sequence substantially identical thereto). In certain embodiments,the FGFR2-BICC1 fusion comprises at least 1, 2, 3, 4, 5, 6, 7, 9, 10,11, 12, 13, 14, 15, 16 or more exons from FGFR2 and at least 1, 2, 3, 4,or more exons from BICC1 (e.g., from the FGFR2 and BICC1 sequences shownin FIGS. 2-3 and 8-9 (SEQ ID NOs:1-2 and 7-8, or a sequencesubstantially identical thereto). In certain embodiments, theBICC1-FGFR2-fusion comprises one or more (or all of) exons 1-2 and exon17 from FGFR2 (e.g., one or more of the exons shown in FIGS. 2-3 and 8-9, or a sequence substantially identical thereto) (e.g., from the FGFR2and BICC1 sequences shown in FIGS. 2-3 and 8-9 (SEQ ID NOs:1-2 and 7-8,or a sequence substantially identical thereto). Additional fusions andexon combinations are disclosed in FIG. 1B.

In another embodiment, the nucleic acid molecule includes a nucleotidesequence that includes a breakpoint, e.g., a breakpoint depicted in FIG.1A, or a sequence substantially identical thereto. In one embodiment,the nucleic acid molecule is complementary to at least a portion of anucleotide sequence disclosed herein, e.g., is capable of hybridizingunder a stringency condition described herein to SEQ ID NO:1 or SEQ IDNO:11 and/or SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:13, or SEQ ID NO:15 or a fragment thereof. In yet other embodiment,the nucleic acid molecule hybridizes to a nucleotide sequence that iscomplementary to at least a portion of a nucleotide sequence disclosedherein, e.g., is capable of hybridizing under a stringency conditiondescribed herein to a nucleotide sequence complementary to SEQ ID NO:1or SEQ ID NO:11 and/or SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:13, or SEQ ID NO:15, or a fragment thereof.

In a related aspect, the invention features nucleic acid constructs thatinclude the FGFR2 nucleic acid molecules described herein. In certainembodiments, the nucleic acid molecules are operatively linked to anative or a heterologous regulatory sequence. Also included are vectorsand host cells that include the FGFR2 nucleic acid molecules describedherein, e.g., vectors and host cells suitable for producing the nucleicacid molecules and polypeptides described herein.

In a related aspect, methods of producing the nucleic acid molecules andpolypeptides described herein are also described.

In another aspect, the invention features nucleic acid molecules thatreduce or inhibit the expression of a nucleic acid molecule that encodesa FGFR2 fusion described herein. Examples of such nucleic acid moleculesinclude, for example, antisense molecules, ribozymes, RNAi, triple helixmolecules that hybridize to a nucleic acid encoding FGFR2, or atranscription regulatory region of FGFR2, and blocks or reduces mRNAexpression of FGFR2.

Nucleic Acid Detection and Capturing Reagents

The invention also features a nucleic acid molecule, e.g., nucleic acidfragment, suitable as probe, primer, bait or library member thatincludes, flanks, hybridizes to, which are useful for identifying, orare otherwise based on, the FGFR2 fusions described herein. In certainembodiments, the probe, primer or bait molecule is an oligonucleotidethat allows capture, detection or isolation of an FGFR2 fusion nucleicacid molecule described herein. The oligonucleotide can comprise anucleotide sequence substantially complementary to a fragment of theFGFR2 fusion nucleic acid molecules described herein. The sequenceidentity between the nucleic acid fragment, e.g., the oligonucleotide,and the target FGFR2 sequence need not be exact, so long as thesequences are sufficiently complementary to allow the capture, detectionor isolation of the target sequence. In one embodiment, the nucleic acidfragment is a probe or primer that includes an oligonucleotide betweenabout 5 and 25, e.g., between 10 and 20, or 10 and 15 nucleotides inlength. In other embodiments, the nucleic acid fragment is a bait thatincludes an oligonucleotide between about 100 to 300 nucleotides, 130and 230 nucleotides, or 150 and 200 nucleotides, in length.

In one embodiment, the nucleic acid fragment can be used to identify orcapture, e.g., by hybridization, an FGFR2 fusion. For example, thenucleic acid fragment can be a probe, a primer, or a bait, for use inidentifying or capturing, e.g., by hybridization, an FGFR2 fusiondescribed herein. In one embodiment, the nucleic acid fragment can beuseful for identifying or capturing an FGFR2 breakpoint, e.g., thenucleotide sequence of: chromosome 10 at the nucleotides depicted inFIG. 1A plus or minus 10, 20, 30, 40 50, 60, 80, 100, 150 nucleotidesand chromosome 4 at the nucleotides depicted in FIG. 1A plus or minus10, 20, 30, 40 50, 60, 80, 100, 150 nucleotides.

In one embodiment, the nucleic acid fragment hybridizes to a nucleotidesequence within a chromosomal rearrangement that creates an in-framefusion of intron 16 of FGFR3 with an intron depicted in FIG. 1A. In oneembodiment, the nucleic acid fragment hybridizes to a nucleotidesequence in the region

In another embodiment, the nucleic acid fragment hybridizes to anucleotide sequence that comprises at least 6, 12, 15, 20, 25, 50, 75,100, 150 or more nucleotides from exon 16 of FGFR2 (e.g., from thenucleotide sequence of FGFR3 preceding the fusion junction with thepartner, e.g., a partner depicted in FIGS. 1A-1C.

In another embodiment, the nucleic acid fragment hybridizes to anucleotide sequence that comprises at least 6, 12, 15, 20, 25, 50, 75,100, 150 or more nucleotides from exon 16 of FGFR2 (e.g., from thenucleotide sequence of FGFR2 preceding the fusion junction with apartner TACC3, KIAA1598, BICC1, PARK2, NOL4 or ZDHHC6.

The probes or primers described herein can be used, for example, forFISH detection or PCR amplification. In one exemplary embodiment wheredetection is based on PCR, amplification of the FGFR2 fusion junctionfusion junction can be performed using a primer or a primer pair, e.g.,for amplifying a sequence flanking the fusion junctions describedherein, e.g., the mutations or the junction of a chromosomalrearrangement described herein, e.g., FGFR2.

In one embodiment, a pair of isolated oligonucleotide primers canamplify a region containing or adjacent to a position in the FGFR2fusion. For example, forward primers can be designed to hybridize to anucleotide sequence within FGFR2 genomic or mRNA sequence, and thereverse primers can be designed to hybridize to a nucleotide sequence ofTACC3, KIAA1598, BICC1, PARK2, NOL4 or ZDHHC6.

In another embodiment, the nucleic acid fragments can be used toidentify, e.g., by hybridization, an FGFR2 fusion. In one embodiment,the nucleic acid fragment hybridizes to a nucleotide sequence thatincludes a fusion junction between the FGFR2 transcript and the partnertranscript.

In other embodiments, the nucleic acid fragment includes a bait thatcomprises a nucleotide sequence that hybridizes to the FGFR2 fusionnucleic acid molecule described herein, and thereby allows the captureor isolation said nucleic acid molecule. In one embodiment, a bait issuitable for solution phase hybridization. In other embodiments, a baitincludes a binding entity, e.g., an affinity tag, that allows captureand separation, e.g., by binding to a binding entity, of a hybrid formedby a bait and a nucleic acid hybridized to the bait.

In other embodiments, the nucleic acid fragment includes a librarymember comprising the FGFR2 nucleic acid molecule described herein. Inone embodiment, the library member includes a rearrangement that resultsin the FGFR2 fusion described herein.

The nucleic acid fragment can be detectably labeled with, e.g., aradiolabel, a fluorescent label, a bioluminescent label, achemiluminescent label, an enzyme label, a binding pair label, or caninclude an affinity tag; a tag, or identifier (e.g., an adaptor, barcodeor other sequence identifier).

Detection Reagents and Detection of Mutations

In another aspect, the invention features a detection reagent, e.g., apurified or an isolated preparation thereof. Detection reagents candistinguish a nucleic acid, or protein sequence, having a breakpoint,e.g., a FGFR2 breakpoint; from a reference sequence (e.g., a breakpointdisclosed herein, e.g., in FIGS. 1A-1C. In one embodiment, the detectionreagent detects (e.g., specifically detects) a FGFR2 fusion nucleic acidor a polypeptide (e.g., distinguishes a wild type TACC3 or another TACC3fusion (or FGFR2) from a FGFR2 nucleic acid (e.g., as described herein).

Detection reagents, e.g., nucleic acid-based detection reagents, can beused to identify mutations in a target nucleic acid, e.g., DNA, e.g.,genomic DNA or cDNA, or RNA, e.g., in a sample, e.g., a sample ofnucleic acid derived from a neoplastic or tumor cell, e.g., amelanocytic neoplasm, melanoma or metastatic cell. Detection reagents,e.g., antibody-based detection reagents, can be used to identifymutations in a target protein, e.g., in a sample, e.g., a sample ofprotein derived from, or produced by, a neoplastic or tumor cell, e.g.,a cholangiocarcinoma or metastatic cell.

Probes

The invention also provides isolated nucleic acid molecules useful asprobes. Such nucleic acid probes can be designed based on the sequenceof a fusion.

Probes based on the sequence of a fusion nucleic acid molecule asdescribed herein can be used to detect transcripts or genomic sequencescorresponding to one or more markers featured in the invention. Theprobe comprises a label group attached thereto, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as part of a test kit for identifying cells or tissues whichexpress the fusion protein such as by measuring levels of a nucleic acidmolecule encoding the protein in a sample of cells from a subject, e.g.,detecting mRNA levels or determining whether a gene encoding the proteinhas been mutated or deleted.

Probes featured in the invention include those that will specificallyhybridize to a gene sequence described in herein. Typically these probesare 12 to 20, e.g., 17 to 20 nucleotides in length (longer for largeinsertions) and have the nucleotide sequence corresponding to the regionof the mutations at their respective nucleotide locations on the genesequence. Such molecules can be labeled according to any technique knownin the art, such as with radiolabels, fluorescent labels, enzymaticlabels, sequence tags, biotin, other ligands, etc. As used herein, aprobe that “specifically hybridizes” to a fusion gene sequence willhybridize under high stringency conditions.

A probe will typically contain one or more of the specific mutationsdescribed herein. Typically, a nucleic acid probe will encompass onlyone mutation. Such molecules may be labeled and can be used asallele-specific probes to detect the mutation of interest.

In one aspect, the invention features a probe or probe set thatspecifically hybridizes to a nucleic acid comprising an inversionresulting in a fusion. In another aspect, the invention features a probeor probe set that specifically hybridizes to a nucleic acid comprising adeletions resulting in a fusion.

Isolated pairs of allele specific oligonucleotide probes are alsoprovided, where the first probe of the pair specifically hybridizes tothe mutant allele, and the second probe of the pair specificallyhybridizes to the wildtype allele. For example, in one exemplary probepair, one probe will recognize the fusion junction in the fusion, andthe other probe will recognize a sequence downstream or upstream of,neither of which includes the fusion junction. These allele-specificprobes are useful in detecting an fusion partner somatic mutation in atumor sample, e.g., cholangiocarcinoma sample.

Primers

The invention also provides isolated nucleic acid molecules useful asprimers.

The term “primer” as used herein refers to a sequence comprising two ormore deoxyribonucleotides or ribonucleotides, e.g., more than three, andmore than eight, or at least 20 nucleotides of a gene described inherein, where the sequence corresponds to a sequence flanking one of themutations or a wild type sequence of a gene identified herein gene.Primers may be used to initiate DNA synthesis via the PCR (polymerasechain reaction) or a sequencing method. Primers featured in theinvention include the sequences recited and complementary sequenceswhich would anneal to the opposite DNA strand of the sample target.Since both strands of DNA are complementary and mirror images of eachother, the same segment of DNA will be amplified.

Primers can be used to sequence a nucleic acid, e.g., an isolatednucleic acid described herein, such as by an NGS method, or to amplify agene described in the Example, such as by PCR. The primers canspecifically hybridize, for example, to the ends of the exons or to theintrons flanking the exons. The amplified segment can then be furtheranalyzed for the presence of the mutation such as by a sequencingmethod. The primers are useful in directing amplification of a targetpolynucleotide prior to sequencing. In another aspect, the inventionfeatures a pair of oligonucleotide primers that amplify a region thatcontains or is adjacent to a fusion junction identified in the Example.Such primers are useful in directing amplification of a target regionthat includes a fusion junction identified herein, e.g., prior tosequencing. The primer typically contains 12 to 20, or 17 to 20, or morenucleotides, although a primer may contain fewer nucleotides.

A primer is typically single stranded, e.g., for use in sequencing oramplification methods, but may be double stranded. If double stranded,the primer may first be treated to separate its strands before beingused to prepare extension products. A primer must be sufficiently longto prime the synthesis of extension products in the presence of theinducing agent for polymerization. The exact length of primer willdepend on many factors, including applications (e.g., amplificationmethod), temperature, buffer, and nucleotide composition. A primertypically contains 12-20 or more nucleotides, although a primer maycontain fewer nucleotides.

Primers are typically designed to be “substantially” complementary toeach strand of a genomic locus to be amplified. Thus, the primers mustbe sufficiently complementary to specifically hybridize with theirrespective strands under conditions which allow the agent forpolymerization to perform. In other words, the primers should havesufficient complementarity with the 5′ and 3′ sequences flanking themutation to hybridize therewith and permit amplification of the genomiclocus.

The term “substantially complementary to” or “substantially thesequence” refers to sequences that hybridize to the sequences providedunder stringent conditions and/or sequences having sufficient homologywith a sequence comprising a fusion junction identified in the Example,or the wildtype counterpart sequence, such that the allele specificoligonucleotides hybridize to the sequence. In one embodiment, asequence is substantially complementary to a fusion junction in aninversion event, e.g., to a fusion junction. “Substantially the same” asit refers to oligonucleotide sequences also refers to the functionalability to hybridize or anneal with sufficient specificity todistinguish between the presence or absence of the mutation. This ismeasurable by the temperature of melting being sufficiently different topermit easy identification of whether the oligonucleotide is binding tothe normal or mutant gene sequence identified in the Example.

In one aspect, the invention features a primer or primer set foramplifying a nucleic acid comprising an inversion resulting in a fusion.In another aspect, the invention features a primer or primer set foramplifying a nucleic acid comprising a deletion resulting in an fusion.

Isolated pairs of allele specific oligonucleotide primer are alsoprovided, where the first primer of the pair specifically hybridizes tothe mutant allele, and the second primer of the pair specificallyhybridizes to a sequence upstream or downstream of a mutation, or afusion junction resulting from, e.g., an inversion, duplication,deletion, insertion or translocation. For example, in one exemplaryprimer pair, one probe will recognize a translocation, such as byhybridizing to a sequence at the fusion junction between the fusionpartner transcripts, and the other primer will recognize a sequenceupstream or downstream of the fusion junction. These allele-specificprimers are useful for amplifying a fusion sequence from a tumor sample,e.g., cholangiocarcinoma. Similarly, in one exemplary primer pair, oneprobe will recognize a fusion, such as by hybridizing to a sequence atthe fusion junction between the transcripts, and the other primer willrecognize a sequence upstream or downstream of the fusion junction.These allele-specific primers are useful for amplifying a fusionsequence from a tumor sample.

In another exemplary primer pair, one primer can recognize antranslocation such as by hybridizing to a sequence at the fusionjunction between the transcripts, and the other primer will recognize asequence upstream or downstream of the fusion junction. Theseallele-specific primers are useful for amplifying a fusion sequence froma cholangiocarcinoma sample.

In addition, an exemplary primer pair can be designed such that oneprimer recognizes an fusion, such as by hybridizing to a sequence at thefusion junction between the transcripts, and the other primer willrecognize a sequence upstream or downstream of the fusion junction.These allele-specific primers are useful for amplifying a fusionsequence from a tumor sample, e.g., a cholangiocarcinoma sample.

Primers can be prepared using any suitable method, such as conventionalphosphotriester and phosphodiester methods or automated embodimentsthereof. In one such automated embodiment, diethylphosphoramidites areused as starting materials and may be synthesized as described byBeaucage, et al., Tetrahedron Letters, 22:1859-1862, (1981). One methodfor synthesizing oligonucleotides on a modified solid support isdescribed in U.S. Pat. No. 4,458,066.

An oligonucleotide probe or primer that hybridizes to a mutant orwildtype allele is said to be the complement of the allele. As usedherein, a probe exhibits “complete complementarity” when everynucleotide of the probe is complementary to the corresponding nucleotideof the allele. Two polynucleotides are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, thepolynucleotides are said to be “complementary” if they can hybridize toone another with sufficient stability to permit them to remain annealedto one another under conventional “high-stringency” conditions.Conventional stringency conditions are known to those skilled in the artand can be found, for example in Molecular Cloning: A Laboratory Manual,3rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N.Irwin, Cold Spring Harbor Laboratory Press, 2000.

Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of aprobe to hybridize to an allele. Thus, in order for a polynucleotide toserve as a primer or probe it need only be sufficiently complementary insequence to be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed. Appropriatestringency conditions which promote DNA hybridization are, for example,6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by awash of 2.0×SSC at 50° C. Such conditions are known to those skilled inthe art and can be found, for example in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989). Salt concentration andtemperature in the wash step can be adjusted to alter hybridizationstringency. For example, conditions may vary from low stringency ofabout 2.0×SSC at 40° C. to moderately stringent conditions of about2.0×SSC at 50° C. to high stringency conditions of about 0.2×SSC at 50°C.

Fusion Proteins and Antibodies

One aspect featured in the invention pertains to purified fusionpolypeptides, and biologically active portions thereof. In oneembodiment, the native fusion polypeptide can be isolated from cells ortissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, fusionpolypeptide is produced by recombinant DNA techniques. Alternative torecombinant expression, a fusion polypeptide can be synthesizedchemically using standard peptide synthesis techniques.

FGFR2 Fusion Polypeptides

In another embodiment, the FGFR2 fusion comprises an amino acid sequenceshown in FIG. 3 (SEQ ID NO:2) or FIG. 13 (SEQ ID NO:12) or a fragmentthereof, and a partner chosen from an amino acid sequence of SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, or SEQ IDNO: 16, or a fragment thereof. In one embodiment, the FGFR2 fusioncomprises an amino acid sequence substantially identical to the aminoacid sequence shown in FIG. 3 (SEQ ID NO:2) or FIG. 13 (SEQ ID NO:12)and SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:14, or SEQ ID NO: 16, or a fragment thereof. In one embodiment, theFGFR2 fusion comprises an amino acid sequence at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, at least 99.5 or greater, identical to the amino acid sequenceshown in FIG. 3 (SEQ ID NO:2) or FIG. 13 (SEQ ID NO:12) and SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, or SEQ IDNO: 16. In one embodiment, the FGFR2 fusion comprises a sequencecontaining at least 10, 20, 50, 100, 500, 600, 700, 800, 900, 1000, ormore amino acids of the amino acid sequence shown in FIG. 3 (SEQ IDNO:2) or FIG. 13 (SEQ ID NO:12); and at least 5, 10, 20, 50, 100, 500,600, 700, 800, 900, 1000, or more amino acids of the amino acid sequenceshown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 14, or SEQ ID NO: 16. In one embodiment, the FGFR2 fusion comprisesan amino acid sequence containing at least 10, 20, 50, 100, 500, 600,700, 800, 900, 1000, or more contiguous amino acids of the amino acidsequence shown in FIG. 3 (SEQ ID NO:2) or FIG. 13 (SEQ ID NO:12); and atleast 5, 10, 20, 50, 100, 500, 600, 700, 800, 900, 1000, or morecontiguous amino acids of the amino acid sequence shown in SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO:16. In one embodiment, the FGFR2 fusion polypeptide includes a FGFR2receptor tyrosine kinase domain or a functional fragment thereof. In anembodiment, the FGFR2 fusion polypeptide comprises sufficient partnersequence, e.g., TACC3, and sufficient FGFR2 sequence such that it haskinase activity, e.g., has elevated activity, e.g., FGFR2 kinaseactivity, as compared with wild type FGFR2, e.g., in a cell of a cancerreferred to herein.

In another aspect, the invention features a FGFR2 fusion polypeptide(e.g., a purified FGFR2 fusion polypeptide), a biologically active orantigenic fragment thereof, as well as reagents (e.g., antibodymolecules that bind to a FGFR2 fusion polypeptide), methods formodulating a FGFR2 polypeptide activity and detection of a FGFR2polypeptide.

In one embodiment, the FGFR2 fusion polypeptide has at least onebiological activity, e.g., an FGFR2 kinase activity. In one embodiment,at least one biological activity of the FGFR2 fusion polypeptide isreduced or inhibited by an anti-cancer drug, e.g., a kinase inhibitor(e.g., a multikinase inhibitor or an FGFR2-specific inhibitor). In oneembodiment, at least one biological activity of the FGFR2 fusionpolypeptide is reduced or inhibited by an FGFR2 kinase inhibitor chosenfrom an inhibitor depicted in Table 2.

In yet other embodiments, the FGFR2 fusion polypeptide is encoded by anucleic acid molecule described herein.

In certain embodiments, the FGFR2 fusion polypeptide comprises one ormore of encoded exons 1-16 from FGFR2 and one or more of encoded exonsof a partner depicted in FIGS. 1A-1C.

In one embodiment, the FGFR2 fusion polypeptide includes a FGFR2tyrosine kinase domain or a functional fragment thereof. In a relatedaspect, the invention features FGFR2 fusion polypeptide or fragmentsoperatively linked to heterologous polypeptides to form fusion proteins.

In another embodiment, the FGFR2 fusion polypeptide or fragment is apeptide, e.g., an immunogenic peptide or protein, that contains a fusionjunction described herein. Such immunogenic peptides or proteins can beused to raise antibodies specific to the fusion protein. In otherembodiments, such immunogenic peptides or proteins can be used forvaccine preparation. The vaccine preparation can include othercomponents, e.g., an adjuvant.

In another aspect, the invention features antibody molecules that bindto a FGFR2 fusion polypeptide or fragment described herein. Inembodiments the antibody can distinguish wild type TACC3, KIAA1598,BICC1 or PARK2, NOL4 or ZDHHC6 (or FGFR2) from FGFR2.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, less than about 20%, less than about 10%, orless than about 5% (by dry weight) of heterologous protein (alsoreferred to herein as a “contaminating protein”). When the protein orbiologically active portion thereof is recombinantly produced, it can besubstantially free of culture medium, i.e., culture medium representsless than about 20%, less than about 10%, or less than about 5% of thevolume of the protein preparation. When the protein is produced bychemical synthesis, it can substantially be free of chemical precursorsor other chemicals, i.e., it is separated from chemical precursors orother chemicals which are involved in the synthesis of the protein.Accordingly such preparations of the protein have less than about 30%,less than about 20%, less than about 10%, less than about 5% (by dryweight) of chemical precursors or compounds other than the polypeptideof interest.

Biologically active portions of a fusion polypeptide includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the fusion protein, whichinclude fewer amino acids than the full length protein, and exhibit atleast one activity of the corresponding full-length protein, e.g., akinase activity. A biologically active portion of a protein featured inthe invention can be a polypeptide which is, for example, 10, 25, 50,100 or more amino acids in length. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of the native form of a polypeptide.

In certain embodiments, the fusion polypeptide has an amino acidsequence of a protein encoded by a nucleic acid molecule disclosedherein. Other useful proteins are substantially identical (e.g., atleast 60, at least 65, at least 70, at least 75, at least 80, at least85, at least 86, at least 87, at least 88, at least 89, at least 90, atleast 91, at least 92, at least 93, at least 94, at least 95, at least96, at least 97, at least 98, at least 99, at least 99.5% or greater) toone of these sequences and retain the functional activity of the proteinof the corresponding full-length protein yet differ in amino acidsequence.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. Another, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules featured inthe invention. BLAST protein searches can be performed with the XBLASTprogram, score=50, word length=3 to obtain amino acid sequenceshomologous to protein molecules featured in the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi nlm nih.gov. Another non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) Comput Appl Biosci, 4:11-7. Suchan algorithm is incorporated into the ALIGN program (version 2.0) whichis part of the GCG sequence alignment software package. When utilizingthe ALIGN program for comparing amino acid sequences, a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused. Yet another useful algorithm for identifying regions of localsequence similarity and alignment is the FASTA algorithm as described inPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. Whenusing the FASTA algorithm for comparing nucleotide or amino acidsequences, a PAM120 weight residue table can, for example, be used witha k-tuple value of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

An isolated fusion polypeptide, or a fragment thereof, can be used as animmunogen to generate antibodies using standard techniques forpolyclonal and monoclonal antibody preparation. The full-length fusionpolypeptide can be used or, alternatively, the invention providesantigenic peptide fragments for use as immunogens. The antigenic peptideof a protein featured in the invention comprises at least 8 (or at least10, at least 15, at least 20, or at least 30 or more) amino acidresidues of the amino acid sequence of one of the polypeptides featuredin the invention, and encompasses an epitope of the protein such that anantibody raised against the peptide forms a specific immune complex witha marker featured in the invention to which the protein corresponds.Exemplary epitopes encompassed by the antigenic peptide are regions thatare located on the surface of the protein, e.g., hydrophilic regions.Hydrophobicity sequence analysis, hydrophilicity sequence analysis, orsimilar analyses can be used to identify hydrophilic regions.

An immunogen typically is used to prepare antibodies by immunizing asuitable (i.e., immunocompetent) subject such as a rabbit, goat, mouse,or other mammal or vertebrate. An appropriate immunogenic preparationcan contain, for example, recombinantly-expressed orchemically-synthesized polypeptide. The preparation can further includean adjuvant, such as Freund's complete or incomplete adjuvant, or asimilar immunostimulatory agent.

Accordingly, another aspect featured in the invention pertains toantibodies directed against a fusion polypeptide. In one embodiment, theantibody molecule specifically binds to fusion, e.g., specifically bindsto an epitope formed by the fusion. In embodiments the antibody candistinguish wild type from fusion.

Another aspect featured in the invention provides antibodies directedagainst a fusion polypeptide are contemplated. In one embodiment, theantibody molecule specifically binds to La fusion, e.g., specificallybinds to an epitope formed by the fusion. In embodiments the antibodycan distinguish wild typefrom the fusion.

The terms “antibody” and “antibody molecule” as used interchangeablyherein refer to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantigen binding site which specifically binds an antigen, such as apolypeptide featured in the invention. A molecule which specificallybinds to a given polypeptide featured in the invention is a moleculewhich binds the polypeptide, but does not substantially bind othermolecules in a sample, e.g., a biological sample, which naturallycontains the polypeptide. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)2 fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies. The term“monoclonal antibody” or “monoclonal antibody composition,” as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a fusion polypeptide as an immunogen.Antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (see Kozbor etal., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Coleet al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., 1985) or trioma techniques. The technology for producinghybridomas is well known (see generally Current Protocols in Immunology,Coligan et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cellsproducing a monoclonal antibody are detected by screening the hybridomaculture supernatants for antibodies that bind the polypeptide ofinterest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with the polypeptide of interest. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication No. WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication No. WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions canbe made using standard recombinant DNA techniques. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described in PCTPublication No. WO 87/02671; European Patent Application 184,187;European Patent Application 171,496; European Patent Application173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Application 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559; Morrison (1985)Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat.No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

Completely human antibodies can be produced using transgenic mice whichare incapable of expressing endogenous immunoglobulin heavy and lightchains genes, but which can express human heavy and light chain genes.For an overview of this technology for producing human antibodies, seeLonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA),can be engaged to provide human antibodies directed against a selectedantigen using technology similar to that described above.

An antibody directed against a fusion polypeptide or a fusionpolypeptide (e.g., a monoclonal antibody) can be used to isolate thepolypeptide by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, such an antibody can be used to detectthe marker (e.g., in a cellular lysate or cell supernatant) in order toevaluate the level and pattern of expression of the marker. Detectioncan be facilitated by coupling the antibody to a detectable substance.Examples of detectable substances include, but are not limited to,various enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include, but are not limited to, horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude, but are not limited to, streptavidin/biotin and avidin/biotin;examples of suitable fluorescent materials include, but are not limitedto, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes, but is not limited to,luminol; examples of bioluminescent materials include, but are notlimited to, luciferase, luciferin, and aequorin, and examples ofsuitable radioactive materials include, but are not limited to, ¹²⁵I,¹³¹I, ³⁵S or ³H.

An antibody directed against a fusion polypeptide can also be useddiagnostically to monitor protein levels in tissues or body fluids(e.g., in a tumor cell-containing body fluid) as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen.

Antigens and Vaccines

Embodiments featured in the invention include preparations, e.g.,antigenic preparations, of the entire fusion or a fragment thereof,e.g., a fragment capable of raising antibodies specific to the fusionprotein, e.g., a fusion junction containing fragment (collectivelyreferred to herein as a fusion specific polypeptides or FSP). Thepreparation can include an adjuvant or other component.

An FSP can be used as an antigen or vaccine. For example, an FSP can beused as an antigen to immunize an animal, e.g., a rodent, e.g., a mouseor rat, rabbit, horse, goat, dog, or non-human primate, to obtainantibodies, e.g., fusion protein specific antibodies. In an embodiment afusion specific antibody molecule is an antibody molecule describedherein, e.g., a polyclonal. In other embodiments a fusion specificantibody molecule is monospecific, e.g., monoclonal, human, humanized,chimeric or other monospecific antibody molecule. A fusion proteinspecific antibody molecule can be used to treat a subject having acholangiocarcinoma.

Embodiments featured in the invention include vaccine preparations thatcomprise an FSP capable of stimulating an immune response in a subject,e.g., by raising, in the subject, antibodies specific to the fusionprotein. The vaccine preparation can include other components, e.g., anadjuvant. The vaccine preparations can be used to treat a subject havingcholangiocarcinoma.

Expression Vectors, Host Cells and Recombinant Cells

In another aspect, the invention includes vectors (e.g., expressionvectors), containing a nucleic acid encoding a fusion polypeptide orencoding an fusion polypeptide as described herein. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked and can include aplasmid, cosmid or viral vector. The vector can be capable of autonomousreplication or it can integrate into a host DNA. Viral vectors include,e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses.

A vector can include a fusion nucleic acid in a form suitable forexpression of the nucleic acid in a host cell. Preferably therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid sequence to be expressed. Theterm “regulatory sequence” includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those which direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorscan be introduced into host cells to thereby produce a fusionpolypeptide, including fusion proteins or polypeptides encoded bynucleic acids as described herein, mutant forms thereof, and the like).

The term “recombinant host cell” (or simply “host cell” or “recombinantcell”), as used herein, is intended to refer to a cell into which arecombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell, but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The recombinant expression vectors can be designed for expression of afusion polypeptide in prokaryotic or eukaryotic cells. For example,polypeptides featured in the invention can be expressed in E. coli,insect cells (e.g., using baculovirus expression vectors), yeast cellsor mammalian cells. Suitable host cells are discussed further inGoeddel, (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, CA. Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRITS (Pharmacia,Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

Purified fusion polypeptides can be used in activity assays (e.g.,direct assays or competitive assays described in detail below), or togenerate antibodies specific for fusion polypeptides.

To maximize recombinant protein expression in E. coli is to express theprotein in a host bacteria with an impaired capacity to proteolyticallycleave the recombinant protein (Gottesman, S., (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego,California 119-128). Another strategy is to alter the nucleic acidsequence of the nucleic acid to be inserted into an expression vector sothat the individual codons for each amino acid are those preferentiallyutilized in E. coli (Wada et al., (1992) Nucleic Acids Res.20:2111-2118). Such alteration of nucleic acid sequences can be carriedout by standard DNA synthesis techniques.

The fusion polypeptide expression vector can be a yeast expressionvector, a vector for expression in insect cells, e.g., a baculovirusexpression vector or a vector suitable for expression in mammaliancells.

When used in mammalian cells, the expression vector's control functionscan be provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40.

In another embodiment, the promoter is an inducible promoter, e.g., apromoter regulated by a steroid hormone, by a polypeptide hormone (e.g.,by means of a signal transduction pathway), or by a heterologouspolypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and“Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy9:983).

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters(Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230:912-1716), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, for example, the murine hox promoters(Kessel and Gruss (1990) Science 249:374-379) and the □-fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule featured in the invention cloned into theexpression vector in an antisense orientation. Regulatory sequences(e.g., viral promoters and/or enhancers) operatively linked to a nucleicacid cloned in the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., fusion nucleic acidmolecule within a recombinant expression vector or a fusion nucleic acidmolecule containing sequences which allow it to homologous recombinationinto a specific site of the host cell's genome.

A host cell can be any prokaryotic or eukaryotic cell. For example, afusion polypeptide can be expressed in bacterial cells (such as E.coli), insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells, e.g., COS-7 cells, CV-1 origin SV40cells; Gluzman (1981) Cell 23:175-182). Other suitable host cells areknown to those skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

A host cell can be used to produce (e.g., express) a fusion polypeptide.Accordingly, the invention further provides methods for producing afusion polypeptide using the host cells. In one embodiment, the methodincludes culturing the host cell (into which a recombinant expressionvector encoding a polypeptide has been introduced) in a suitable mediumsuch that the fusion polypeptide is produced. In another embodiment, themethod further includes isolating a fusion polypeptide from the mediumor the host cell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a fusion transgene, or whichotherwise misexpress fusion. In another aspect, the invention features,a cell or purified preparation of cells which include a fusiontransgene, or which otherwise misexpress a fusion.

The cell preparation can consist of human or non-human cells, e.g.,rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. Inembodiments, the cell or cells include a fusion transgene, e.g., aheterologous form of a fusion, e.g., a gene derived from humans (in thecase of a non-human cell) or a fusion transgene, e.g., a heterologousform of a fusion. The fusion transgene can be misexpressed, e.g.,overexpressed or underexpressed. In other preferred embodiments, thecell or cells include a gene that mis-expresses an endogenous fusion,e.g., a gene the expression of which is disrupted, e.g., a knockout.Such cells can serve as a model for studying disorders that are relatedto mutated or mis-expressed fusion alleles (e.g., cancers) or for use indrug screening, as described herein.

Screening Methods

In another aspect, the invention features a method, or assay, forscreening for agents that modulate, e.g., inhibit, the expression oractivity of a fusion, described herein. The method includes contacting afusion, or a cell expressing a fusion, with a candidate agent; anddetecting a change in a parameter associated with a fusion, e.g., achange in the expression or an activity of the fusion. The method can,optionally, include comparing the treated parameter to a referencevalue, e.g., a control sample (e.g., comparing a parameter obtained froma sample with the candidate agent to a parameter obtained from a samplewithout the candidate agent). In one embodiment, if a decrease inexpression or activity of the fusion is detected, the candidate agent isidentified as an inhibitor. In another embodiment, if an increase inexpression or activity of the fusion is detected, the candidate agent isidentified as an activator. In certain embodiments, the fusion is anucleic acid molecule or a polypeptide as described herein.

In one embodiment, the contacting step is effected in a cell-freesystem, e.g., a cell lysate or in a reconstituted system. In otherembodiments, the contacting step is effected in a cell in culture, e.g.,a cell expressing a fusion (e.g., a mammalian cell, a tumor cell or cellline, a recombinant cell). In yet other embodiments, the contacting stepis effected in a cell in vivo.

Exemplary parameters evaluated include one or more of:

-   -   (i) a change in binding activity, e.g., direct binding of the        candidate agent to a fusion polypeptide; a binding competition        between a known ligand and the candidate agent to a fusion        polypeptide;    -   (ii) a change in kinase activity, e.g., phosphorylation levels        of a fusion polypeptide (e.g., an increased or decreased        autophosphorylation); or a change in phosphorylation of a target        of fusion. In certain embodiments, a change in kinase activity,        e.g., phosphorylation, is detected by any of Western blot, mass        spectrometry, immunoprecipitation, immunohistochemistry,        immunomagnetic beads, among others;    -   (iii) a change in an activity of a cell containing a fusion        (e.g., a tumor cell or a recombinant cell), e.g., a change in        proliferation, morphology or tumorigenicity of the cell;    -   (iv) a change in tumor present in an animal subject, e.g., size,        appearance, proliferation, of the tumor; or    -   (v) a change in the level, e.g., expression level, of a fusion        polypeptide or nucleic acid molecule.

In one embodiment, a change in a cell free assay in the presence of acandidate agent is evaluated. For example, an activity of a Fusion, orinteraction of a Fusion with a downstream ligand can be detected. In oneembodiment, a Fusion polypeptide is contacted with a ligand, e.g., insolution, and a candidate agent is monitored for an ability to modulate,e.g., inhibit, an interaction, e.g., binding, between the Fusionpolypeptide and the ligand. In one exemplary assay, purified Fusionprotein is contacted with a ligand, e.g., in solution, and a candidateagent is monitored for an ability to inhibit interaction of the fusionprotein with the ligand, or to inhibit phosphorylation of the ligand bythe fusion protein. An effect on an interaction between the fusionprotein and a ligand can be monitored by methods known in the art, suchas by absorbance, and an effect on phosphorylation of the ligand can beassayed, e.g., by Western blot, immunoprecipitation, or immunomagneticbeads.

In other embodiments, a change in an activity of a cell is detected in acell in culture, e.g., a cell expressing a Fusion (e.g., a mammaliancell, a tumor cell or cell line, a recombinant cell). In one embodiment,the cell is a recombinant cell that is modified to express a Fusionnucleic acid, e.g., is a recombinant cell transfected with a Fusionnucleic acid. The transfected cell can show a change in response to theexpressed K Fusion, e.g., increased proliferation, changes inmorphology, increased tumorigenicity, and/or acquired a transformedphenotype. A change in any of the activities of the cell, e.g., therecombinant cell, in the presence of the candidate agent can bedetected. For example, a decrease in one or more of: proliferation,tumorigenicity, transformed morphology, in the presence of the candidateagent can be indicative of an inhibitor of a Fusion. In otherembodiments, a change in binding activity or phosphorylation asdescribed herein is detected.

In an exemplary cell-based assay, a nucleic acid comprising a Fusion canbe expressed in a cell, such as a cell (e.g., a mammalian cell) inculture. The cell containing a nucleic acid expressing the Fusion can becontacted with a candidate agent, and the cell is monitored for aneffect of the candidate agent. A candidate agent that causes decreasedcell proliferation or cell death can be determined to be a candidate fortreating a tumor (e.g., a cancer) that carries a Fusion.

In one embodiment, a cell containing a nucleic acid expressing a Fusioncan be monitored for expression of the Fusion protein. Proteinexpression can be monitored by methods known in the art, such as by,e.g., mass spectrometry (e.g., tandem mass spectrometry), a reporterassay (e.g., a fluorescence-based assay), Western blot, andimmunohistochemistry. By one method, decreased expression is detected. Acandidate agent that causes decreased expression of the Fusion proteinas compared to a cell that does not contain the nucleic acid fusion canbe determined to be a candidate for treating a tumor (e.g., a cancer)that carries a Fusion.

In yet other embodiment, a change in a tumor present in an animalsubject (e.g., an in vivo animal model) is detected. In one embodiment,the animal model is a tumor containing animal or a xenograft comprisingcells expressing a Fusion (e.g., tumorigenic cells expressing a Fusion).The candidate agent can be administered to the animal subject and achange in the tumor is detected. In one embodiment, the change in thetumor includes one or more of a tumor growth, tumor size, tumor burden,survival, is evaluated. A decrease in one or more of tumor growth, tumorsize, tumor burden, or an increased survival is indicative that thecandidate agent is an inhibitor.

In one exemplary animal model, a xenograft is created by injecting cellsinto mouse. A candidate agent is administered to the mouse, e.g., byinjection (such as subcutaneous, intraperitoneal, or tail veininjection, or by injection directly into the tumor) or oral delivery,and the tumor is observed to determine an effect of the candidateanti-cancer agent. The health of the animal is also monitored, such asto determine if an animal treated with a candidate agent surviveslonger. A candidate agent that causes growth of the tumor to slow orstop, or causes the tumor to shrink in size, or causes decreased tumorburden, or increases survival time, can be considered to be a candidatefor treating a tumor (e.g., a cancer) that carries a Fusion.

In another exemplary animal assay, cells expressing a Fusion areinjected into the tail vein, e.g., of a mouse, to induce metastasis. Acandidate agent is administered to the mouse, e.g., by injection (suchas subcutaneous, intraperitoneal, or tail vein injection, or byinjection directly into the tumor) or oral delivery, and the tumor isobserved to determine an effect of the candidate anti-cancer agent. Acandidate agent that inhibits or prevents or reduces metastasis, orincreases survival time, can be considered to be a candidate fortreating a tumor (e.g., a cancer) that carries a Fusion.

Cell proliferation can be measured by methods known in the art, such asPCNA (Proliferating cell nuclear antigen) assay, 5-bromodeoxyuridine(BrdUrd) incorporation, Ki-67 assay, mitochondrial respiration, orpropidium iodide staining. Cells can also be measured for apoptosis,such as by use of a TUNEL (Terminal Deoxynucleotide Transferase dUTPNick End Labeling) assay. Cells can also be assayed for presence ofangiogenesis using methods known in the art, such as by measuringendothelial tube formation or by measuring the growth of blood vesselsfrom subcutaneous tissue, such as into a solid gel of basement membrane.

In other embodiments, a change in expression of a Fusion can bemonitored by detecting the nucleic acid or protein levels, e.g., usingthe methods described herein.

In certain embodiments, the screening methods described herein can berepeated and/or combined. In one embodiment, a candidate agent that isevaluated in a cell-free or cell-based described herein can be furthertested in an animal subject.

In one embodiment, the candidate agent is identified and re-tested inthe same or a different assay. For example, a test compound isidentified in an in vitro or cell-free system, and re-tested in ananimal model or a cell-based assay. Any order or combination of assayscan be used. For example, a high throughput assay can be used incombination with an animal model or tissue culture.

Candidate agents suitable for use in the screening assays describedherein include, e.g., small molecule compounds, nucleic acids (e.g.,siRNA, aptamers, short hairpin RNAs, antisense oligonucleotides,ribozymes, antagomirs, microRNA mimics or DNA, e.g., for gene therapy)or polypeptides, e.g., antibodies (e.g., full length antibodies orantigen-binding fragments thereof, Fab fragments, or scFv fragments).The candidate anti-cancer agents can be obtained from a library (e.g., acommercial library), or can be rationally designed, such as to target anactive site in a functional domain of fusion partner.

In other embodiments, the method, or assay, includes providing a stepbased on proximity-dependent signal generation, e.g., a two-hybrid assaythat includes a first fusion protein (e.g., a Fusion protein), and asecond fusion protein (e.g., a ligand), contacting the two-hybrid assaywith a test compound, under conditions wherein said two hybrid assaydetects a change in the formation and/or stability of the complex, e.g.,the formation of the complex initiates transcription activation of areporter gene.

In one non-limiting example, the three-dimensional structure of theactive site of Fusion is determined by crystallizing the complex formedby the Fusion and a known inhibitor. Rational drug design is then usedto identify new test agents by making alterations in the structure of aknown inhibitor or by designing small molecule compounds that bind tothe active site of the Fusion.

The candidate agents can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; peptoid libraries (libraries of molecules havingthe functionalities of peptides, but with a novel, non-peptide backbonewhich are resistant to enzymatic degradation but which neverthelessremain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med.Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means known in the art (e.g., using afluorimeter).

In another embodiment, determining the ability of the Fusion protein tobind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalwhich can be used as an indication of real-time reactions betweenbiological molecules.

Nucleic Acid Inhibitors

In yet another embodiment, the Fusion inhibitor inhibits the expressionof nucleic acid encoding the fusion. Examples of such fusion inhibitorsinclude nucleic acid molecules, for example, antisense molecules,ribozymes, siRNA, triple helix molecules that hybridize to a nucleicacid encoding a Fusion, or a transcription regulatory region, and blocksor reduces mRNA expression of the fusion.

In one embodiment, the nucleic acid antagonist is a siRNA that targetsmRNA encoding a Fusion. Other types of antagonistic nucleic acids canalso be used, e.g., a dsRNA, a ribozyme, a triple-helix former, or anantisense nucleic acid. Accordingly, isolated nucleic acid moleculesthat are nucleic acid inhibitors, e.g., antisense, RNAi, to aFusion-encoding nucleic acid molecule are provided.

An “antisense” nucleic acid can include a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. The antisense nucleic acid can becomplementary to an entire fusion coding strand, or to only a portionthereof. In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding fusion (e.g., the 5′ and 3′ untranslated regions).Anti-sense agents can include, for example, from about 8 to about 80nucleobases (i.e., from about 8 to about 80 nucleotides), e.g., about 8to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression. Anti-sense compounds can include a stretchof at least eight consecutive nucleobases that are complementary to asequence in the target gene. An oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA can interfere withone or more of the normal functions of mRNA. The functions of mRNA to beinterfered with include all key functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.Binding of specific protein(s) to the RNA may also be interfered with byantisense oligonucleotide hybridization to the RNA.

Exemplary antisense compounds include DNA or RNA sequences thatspecifically hybridize to the target nucleic acid, e.g., the mRNAencoding Fusion. The complementary region can extend for between about 8to about 80 nucleobases. The compounds can include one or more modifiednucleobases. Modified nucleobases are known in the art. Descriptions ofmodified nucleic acid agents are also available. See, e.g., U.S. Pat.Nos. 4,987,071; 5,116,742; and U.S. Pat. No. 5,093,246; Woolf et al.(1992) Proc Natl Acad Sci USA; Antisense RNA and DNA, D. A. Melton, Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988);89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C.(1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher (1992) Bioassays 14:807-15.

The antisense nucleic acid molecules are typically administered to asubject (e.g., by direct injection at a tissue site), or generated insitu such that they hybridize with or bind to cellular mRNA and/orgenomic DNA encoding a fusion to thereby inhibit expression of theprotein, e.g., by inhibiting transcription and/or translation.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then be administered systemically. Forsystemic administration, antisense molecules can be modified such thatthey specifically bind to receptors or antigens expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies which bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing the vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule is anα-anomeric nucleic acid molecule. An α-anomeric nucleic acid moleculeforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

siRNAs are small double stranded RNAs (dsRNAs) that optionally includeoverhangs. For example, the duplex region of an siRNA is about 18 to 25nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotidesin length. Typically, the siRNA sequences are exactly complementary tothe target mRNA. dsRNAs and siRNAs in particular can be used to silencegene expression in mammalian cells (e.g., human cells). siRNAs alsoinclude short hairpin RNAs (shRNAs) with 29-base-pair stems and2-nucleotide 3′ overhangs. See, e.g., Clemens et al. (2000) Proc. Natl.Acad. Sci. USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA98:14428-14433; Elbashir et al. (2001) Nature. 411:494-8; Yang et al.(2002) Proc. Natl. Acad. Sci. USA 99:9942-17947; Siolas et al. (2005),Nat. Biotechnol. 23(2):227-31; 20040086884; U.S. 20030166282;20030143204; 20040038278; and 20030224432.

In still another embodiment, an antisense nucleic acid featured in theinvention is a ribozyme. A ribozyme having specificity for aFusion-encoding nucleic acid can include one or more sequencescomplementary to the nucleotide sequence of a fusion cDNA disclosedherein (i.e., SEQ ID NO:6), and a sequence having known catalyticsequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 orHaselhoff and Gerlach (1988) Nature 334:585-591). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a Fusion-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, fusion mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418.

Inhibition of a Fusion gene can be accomplished by targeting nucleotidesequences complementary to the regulatory region of the fusion to formtriple helical structures that prevent transcription of the Fusion genein target cells. See generally, Helene, C. (1991) Anticancer Drug Des.6:569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher,L. J. (1992) Bioassays 14:807-15. The potential sequences that can betargeted for triple helix formation can be increased by creating aso-called “switchback” nucleic acid molecule. Switchback molecules aresynthesized in an alternating 5′-3′, 3′-5′ manner, such that they basepair with first one strand of a duplex and then the other, eliminatingthe necessity for a sizeable stretch of either purines or pyrimidines tobe present on one strand of a duplex.

The invention also provides detectably labeled oligonucleotide primerand probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

A fusion nucleic acid molecule can be modified at the base moiety, sugarmoiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For non-limiting examplesof synthetic oligonucleotides with modifications see Toulmé (2001)Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44.Such phosphoramidite oligonucleotides can be effective antisense agents.

For example, the deoxyribose phosphate backbone of the nucleic acidmolecules can be modified to generate peptide nucleic acids (see HyrupB. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As usedherein, the terms “peptide nucleic acid” or “PNA” refers to a nucleicacid mimic, e.g., a DNA mimic, in which the deoxyribose phosphatebackbone is replaced by a pseudopeptide backbone and only the fournatural nucleobases are retained. The neutral backbone of a PNA canallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in HyrupB. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci.93: 14670-675.

PNAs of Fusion nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of fusion nucleic acid molecules can also be used inthe analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., 51 nucleases (Hyrup B. etal. (1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652;WO88/09810) or the blood-brain barrier (see, e.g., WO 89/10134). Inaddition, oligonucleotides can be modified with hybridization-triggeredcleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976)or intercalating agents (See, e.g., Zon (1988) Pharm. Res. 5:539-549).To this end, the oligonucleotide may be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

In some embodiments, a nucleic acid inhibitor described herein isprovided for the inhibition of expression of an fusion nucleic acid invitro.

Evaluation of Subjects

Subjects, e.g., patients, can be evaluated for the presence of a fusion.A patient can be evaluated, for example, by determining the genomicsequence of the patient, e.g., by an NGS method. Alternatively, or inaddition, evaluation of a patient can include directly assaying for thepresence of a fusion in the patient, such as by an assay to detect afusion nucleic acid (e.g., DNA or RNA), such as by, Southern blot,Northern blot, or RT-PCR, e.g., qRT-PCR. Alternatively, or in addition,a patient can be evaluated for the presence of a protein fusion, such asby immunohistochemistry, Western blot, immunoprecipitation, orimmunomagnetic bead assay.

Evaluation of a patient can also include a cytogenetic assay, such as byfluorescence in situ hybridization (FISH), to identify the chromosomalrearrangement resulting in the fusion. For example, to perform FISH, atleast a first probe tagged with a first detectable label can be designedto target one fusion partner, and at least a second probe tagged with asecond detectable label can be designed to target the other fusionpartner. The at least one first probe and the at least one second probewill be closer together in patients who carry the fusion than inpatients who do not carry the Fusion. Additional methods for fusiondetection are provided below.

In one aspect, the results of a clinical trial, e.g., a successful orunsuccessful clinical trial, can be repurposed to identify agents thattarget a fusion. By one exemplary method, a candidate agent used in aclinical trial can be reevaluated to determine if the agent in the trialtargets a fusion, or is effective to treat a tumor containing aparticular fusion. For example, subjects who participated in a clinicaltrial for an agent, such as a kinase inhibitor, can be identified.Patients who experienced an improvement in symptoms, e.g., cancer (e.g.,lung cancer) symptoms, such as decreased tumor size, or decreased rateof tumor growth, can be evaluated for the presence of a Fusion. Patientswho did not experience an improvement in cancer symptoms can also beevaluated for the presence of a Fusion. Where patients carrying a Fusionare found to have been more likely to respond to the test agent thanpatients who did not carry such a fusion, then the agent is determinedto be an appropriate treatment option for a patient carrying the fusion.

“Reevaluation” of patients can include, for example, determining thegenomic sequence of the patients, or a subset of the clinical trialpatients, e.g., by an NGS method. Alternatively, or in addition,reevaluation of the patients can include directly assaying for thepresence of a Fusion in the patient, such as by an assay to detect afusion nucleic acid (e.g., RNA), such as by RT-PCR, e.g., qRT-PCR.Alternatively, or in addition, a patient can be evaluated for thepresence of a protein fusion, such as by immunohistochemistry, Westernblot, immunoprecipitation, or immunomagnetic bead assay.

Methods for Detection of Fusion Nucleic Acids and Polypeptides

Methods for evaluating a fusion gene, mutations and/or gene products areknown to those of skill in the art. In one embodiment, the fusion isdetected in a nucleic acid molecule by a method chosen from one or moreof: nucleic acid hybridization assay, amplification-based assays (e.g.,polymerase chain reaction (PCR)), PCR-RFLP assay, real-time PCR,sequencing, screening analysis (including metaphase cytogenetic analysisby standard karyotype methods, FISH (e.g., break away FISH), spectralkaryotyping or MFISH, comparative genomic hybridization), in situhybridization, SSP, HPLC or mass-spectrometric genotyping.

Additional exemplary methods include, traditional “direct probe” methodssuch as Southern blots or in situ hybridization (e.g., fluorescence insitu hybridization (FISH) and FISH plus SKY), and “comparative probe”methods such as comparative genomic hybridization (CGH), e.g.,cDNA-based or oligonucleotide-based CGH, can be used. The methods can beused in a wide variety of formats including, but not limited to,substrate (e.g., membrane or glass) bound methods or array-basedapproaches.

In certain embodiments, the evaluation methods include theprobes/primers described herein.

In one embodiment, probes/primers can be designed to detect a fusion ora reciprocal thereof. These probes/primers are suitable, e.g., for FISHor PCR amplification. In one embodiment, FISH analysis is used toidentify the chromosomal rearrangement resulting in the fusions asdescribed above. In one approach, a variation of a FISH assay, e.g.,“break-away FISH”, is used to evaluate a patient. Other variations ofthe FISH method known in the art are suitable for evaluating a patient.

Probes are used that contain DNA segments that are essentiallycomplementary to DNA base sequences existing in different portions ofchromosomes. Examples of probes useful according to the invention, andlabeling and hybridization of probes to samples are described in twoU.S. patents to Vysis, Inc. U.S. Pat. Nos. 5,491,224 and 6,277,569 toBittner, et al.

Additional protocols for FISH detection are described below.

Chromosomal probes are typically about 50 to about 10⁵ nucleotides inlength. Longer probes typically comprise smaller fragments of about 100to about 500 nucleotides in length. Probes that hybridize withcentromeric DNA and locus-specific DNA are available commercially, forexample, from Vysis, Inc. (Downers Grove, Ill.), Molecular Probes, Inc.(Eugene, Oreg.) or from Cytocell (Oxfordshire, UK).

Alternatively, probes can be made non-commercially from chromosomal orgenomic DNA through standard techniques. For example, sources of DNAthat can be used include genomic DNA, cloned DNA sequences, somatic cellhybrids that contain one, or a part of one, chromosome (e.g., humanchromosome) along with the normal chromosome complement of the host, andchromosomes purified by flow cytometry or microdissection. The region ofinterest can be isolated through cloning, or by site-specificamplification via the polymerase chain reaction (PCR). See, for example,Nath and Johnson, Biotechnic Histochem., 1998, 73(1):6-22, Wheeless etal., Cytometry 1994, 17:319-326, and U.S. Pat. No. 5,491,224.

The probes to be used hybridize to a specific region of a chromosome todetermine whether a cytogenetic abnormality is present in this region.One type of cytogenetic abnormality is a deletion. Although deletionscan be of one or more entire chromosomes, deletions normally involveloss of part of one or more chromosomes. If the entire region of achromosome that is contained in a probe is deleted from a cell,hybridization of that probe to the DNA from the cell will normally notoccur and no signal will be present on that chromosome. If the region ofa chromosome that is partially contained within a probe is deleted froma cell, hybridization of that probe to the DNA from the cell can stilloccur, but less of a signal can be present. For example, the loss of asignal is compared to probe hybridization to DNA from control cells thatdo not contain the genetic abnormalities which the probes are intendedto detect. In some embodiments, at least 1, 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, ormore cells are enumerated for presence of the cytogenetic abnormality.

Cytogenetic abnormalities to be detected can include, but are notlimited to, non-reciprocal translocations, balanced translocations,intra-chromosomal inversions, point mutations, deletions, gene copynumber changes, gene expression level changes, and germ line mutations.In particular, one type of cytogenetic abnormality is a duplication.Duplications can be of entire chromosomes, or of regions smaller than anentire chromosome. If the region of a chromosome that is contained in aprobe is duplicated in a cell, hybridization of that probe to the DNAfrom the cell will normally produce at least one additional signal ascompared to the number of signals present in control cells with noabnormality of the chromosomal region contained in the probe.

Chromosomal probes are labeled so that the chromosomal region to whichthey hybridize can be detected. Probes typically are directly labeledwith a fluorophore, an organic molecule that fluoresces after absorbinglight of lower wavelength/higher energy. The fluorophore allows theprobe to be visualized without a secondary detection molecule. Aftercovalently attaching a fluorophore to a nucleotide, the nucleotide canbe directly incorporated into the probe with standard techniques such asnick translation, random priming, and PCR labeling. Alternatively,deoxycytidine nucleotides within the probe can be transaminated with alinker. The fluorophore then is covalently attached to the transaminateddeoxycytidine nucleotides. See, U.S. Pat. No. 5,491,224.

U.S. Pat. No. 5,491,224 describes probe labeling as a number of thecytosine residues having a fluorescent label covalently bonded thereto.The number of fluorescently labeled cytosine bases is sufficient togenerate a detectable fluorescent signal while the individual so labeledDNA segments essentially retain their specific complementary binding(hybridizing) properties with respect to the chromosome or chromosomeregion to be detected. Such probes are made by taking the unlabeled DNAprobe segment, transaminating with a linking group a number ofdeoxycytidine nucleotides in the segment, covalently bonding afluorescent label to at least a portion of the transaminateddeoxycytidine bases.

Probes can also be labeled by nick translation, random primer labelingor PCR labeling. Labeling is done using either fluorescent (direct)- orhaptene (indirect)-labeled nucleotides. Representative, non-limitingexamples of labels include: AMCA-6-dUTP, CascadeBlue-4-dUTP,Fluorescein-12-dUTP, Rhodamine-6-dUTP, TexasRed-6-dUTP, Cy3-6-dUTP,Cy5-dUTP, Biotin(BIO)-11-dUTP, Digoxygenin(DIG)-11-dUTP or Dinitrophenyl(DNP)-11-dUTP.

Probes also can be indirectly labeled with biotin or digoxygenin, orlabeled with radioactive isotopes such as 32p and 0.3H, althoughsecondary detection molecules or further processing then is required tovisualize the probes. For example, a probe labeled with biotin can bedetected by avidin conjugated to a detectable marker. For example,avidin can be conjugated to an enzymatic marker such as alkalinephosphatase or horseradish peroxidase. Enzymatic markers can be detectedin standard colorimetric reactions using a substrate and/or a catalystfor the enzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

Probes can also be prepared such that a fluorescent or other label isnot part of the DNA before or during the hybridization, and is addedafter hybridization to detect the probe hybridized to a chromosome. Forexample, probes can be used that have antigenic molecules incorporatedinto the DNA. After hybridization, these antigenic molecules aredetected using specific antibodies reactive with the antigenicmolecules. Such antibodies can themselves incorporate a fluorochrome, orcan be detected using a second antibody with a bound fluorochrome.

However treated or modified, the probe DNA is commonly purified in orderto remove unreacted, residual products (e.g., fluorochrome molecules notincorporated into the DNA) before use in hybridization.

Prior to hybridization, chromosomal probes are denatured according tomethods well known in the art. Probes can be hybridized or annealed tothe chromosomal DNA under hybridizing conditions. “Hybridizingconditions” are conditions that facilitate annealing between a probe andtarget chromosomal DNA. Since annealing of different probes will varydepending on probe length, base concentration and the like, annealing isfacilitated by varying probe concentration, hybridization temperature,salt concentration and other factors well known in the art.

Hybridization conditions are facilitated by varying the concentrations,base compositions, complexities, and lengths of the probes, as well assalt concentrations, temperatures, and length of incubation. Forexample, in situ hybridizations are typically performed in hybridizationbuffer containing 1-2×SSC, 50-65% formamide and blocking DNA to suppressnon-specific hybridization. In general, hybridization conditions, asdescribed above, include temperatures of about 25° C. to about 55° C.,and incubation lengths of about 0.5 hours to about 96 hours.

Non-specific binding of chromosomal probes to DNA outside of the targetregion can be removed by a series of washes. Temperature andconcentration of salt in each wash are varied to control stringency ofthe washes. For example, for high stringency conditions, washes can becarried out at about 65° C. to about 80° C., using 0.2× to about 2×SSC,and about 0.1% to about 1% of a non-ionic detergent such as Nonidet P-40(NP40). Stringency can be lowered by decreasing the temperature of thewashes or by increasing the concentration of salt in the washes. In someapplications it is necessary to block the hybridization capacity ofrepetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-I DNA is used to block non-specific hybridization. Afterwashing, the slide is allowed to drain and air dry, then mountingmedium, a counterstain such as DAPI, and a coverslip are applied to theslide. Slides can be viewed immediately or stored at −20° C. beforeexamination.

For fluorescent probes used in fluorescence in situ hybridization (FISH)techniques, fluorescence can be viewed with a fluorescence microscopeequipped with an appropriate filter for each fluorophore, or by usingdual or triple band-pass filter sets to observe multiple fluorophores.See, for example, U.S. Pat. No. 5,776,688. Alternatively, techniquessuch as flow cytometry can be used to examine the hybridization patternof the chromosomal probes.

In CGH methods, a first collection of nucleic acids (e.g., from asample, e.g., a possible tumor) is labeled with a first label, while asecond collection of nucleic acids (e.g., a control, e.g., from ahealthy cell/tissue) is labeled with a second label. The ratio ofhybridization of the nucleic acids is determined by the ratio of the two(first and second) labels binding to each fiber in the array. Wherethere are chromosomal deletions or multiplications, differences in theratio of the signals from the two labels will be detected and the ratiowill provide a measure of the copy number. Array-based CGH can also beperformed with single-color labeling (as opposed to labeling the controland the possible tumor sample with two different dyes and mixing themprior to hybridization, which will yield a ratio due to competitivehybridization of probes on the arrays). In single color CGH, the controlis labeled and hybridized to one array and absolute signals are read,and the possible tumor sample is labeled and hybridized to a secondarray (with identical content) and absolute signals are read. Copynumber difference is calculated based on absolute signals from the twoarrays.

Hybridization protocols suitable for use with the methods featured inthe invention are described, e.g., in Albertson (1984) EMBO J. 3:1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPOPub. No. 430,402; Methods in Molecular Biology, Vol. 33: In situHybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994),etc. In one embodiment, the hybridization protocol of Pinkel, et al.(1998) Nature Genetics 20: 207-211, or of Kallioniemi (1992) Proc. NatlAcad Sci USA 89:5321-5325 (1992) is used. Array-based CGH is describedin U.S. Pat. No. 6,455,258, the contents of each of which areincorporated herein by reference.

In still another embodiment, amplification-based assays can be used tomeasure presence/absence and copy number. In such amplification-basedassays, the nucleic acid sequences act as a template in an amplificationreaction (e.g., Polymerase Chain Reaction (PCR). In a quantitativeamplification, the amount of amplification product will be proportionalto the amount of template in the original sample. Comparison toappropriate controls, e.g., healthy tissue, provides a measure of thecopy number.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that can be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis, et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). Measurement of DNA copy numberat microsatellite loci using quantitative PCR analysis is described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleicacid sequence for the genes is sufficient to enable one of skill in theart to routinely select primers to amplify any portion of the gene.Fluorogenic quantitative PCR can also be used. In fluorogenicquantitative PCR, quantitation is based on amount of fluorescencesignals, e.g., TaqMan and sybr green.

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren, et al. (1988) Science 241:1077, and Barringer et al. (1990)Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

Nucleic Acid Samples

A variety of tissue samples can be the source of the nucleic acidsamples used in the present methods. Genomic or subgenomic DNA fragmentscan be isolated from a subject's sample (e.g., a tumor sample, a normaladjacent tissue (NAT), a blood sample or any normal control)). Incertain embodiments, the tissue sample is preserved as a frozen sampleor as formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE)tissue preparation. For example, the sample can be embedded in a matrix,e.g., an FFPE block or a frozen sample. The isolating step can includeflow-sorting of individual chromosomes; and/or micro-dissecting asubject's sample (e.g., a tumor sample, a NAT, a blood sample).

Protocols for DNA isolation from a tissue sample are known in the art.Additional methods to isolate nucleic acids (e.g., DNA) fromformaldehyde- or paraformaldehyde-fixed, paraffin-embedded (FFPE)tissues are disclosed, e.g., in Cronin M. et al., (2004) Am J Pathol.164(1):35-42; Masuda N. et al., (1999) Nucleic Acids Res.27(22):4436-4443; Specht K. et al., (2001) Am J Pathol. 158(2):419-429,Ambion RecoverAll™ Total Nucleic Acid Isolation Protocol (Ambion, Cat.No. AM1975, September 2008), and QIAamp® DNA FFPE Tissue Handbook(Qiagen, Cat. No. 37625, October 2007). RecoverAll™ Total Nucleic AcidIsolation Kit uses xylene at elevated temperatures to solubilizeparaffin-embedded samples and a glass-fiber filter to capture nucleicacids. QIAamp® DNA FFPE Tissue Kit uses QIAamp® DNA Micro technology forpurification of genomic and mitochondrial DNA.

The isolated nucleic acid samples (e.g., genomic DNA samples) can befragmented or sheared by practicing routine techniques. For example,genomic DNA can be fragmented by physical shearing methods, enzymaticcleavage methods, chemical cleavage methods, and other methods wellknown to those skilled in the art. The nucleic acid library can containall or substantially all of the complexity of the genome. The term“substantially all” in this context refers to the possibility that therecan in practice be some unwanted loss of genome complexity during theinitial steps of the procedure. The methods described herein also areuseful in cases where the nucleic acid library is a portion of thegenome, i.e., where the complexity of the genome is reduced by design.In some embodiments, any selected portion of the genome can be used withthe methods described herein. In certain embodiments, the entire exomeor a subset thereof is isolated.

Methods can further include isolating a nucleic acid sample to provide alibrary (e.g., a nucleic acid library). In certain embodiments, thenucleic acid sample includes whole genomic, subgenomic fragments, orboth. The isolated nucleic acid samples can be used to prepare nucleicacid libraries. Thus, in one embodiment, the methods featured in theinvention further include isolating a nucleic acid sample to provide alibrary (e.g., a nucleic acid library as described herein). Protocolsfor isolating and preparing libraries from whole genomic or subgenomicfragments are known in the art (e.g., Illumina's genomic DNA samplepreparation kit). In certain embodiments, the genomic or subgenomic DNAfragment is isolated from a subject's sample (e.g., a tumor sample, anormal adjacent tissue (NAT), a blood sample or any normal control)). Inone embodiment, the sample (e.g., the tumor or NAT sample) is apreserved. For example, the sample is embedded in a matrix, e.g., anFFPE block or a frozen sample. In certain embodiments, the isolatingstep includes flow-sorting of individual chromosomes; and/ormicrodissecting a subject's sample (e.g., a tumor sample, a NAT, a bloodsample). In certain embodiments, the nucleic acid sample used togenerate the nucleic acid library is less than 5, less than 1 microgram,less than 500 ng, less than 200 ng, less than 100 ng, less than 50 ng orless than 20 ng (e.g., 10 ng or less).

In still other embodiments, the nucleic acid sample used to generate thelibrary includes RNA or cDNA derived from RNA. In some embodiments, theRNA includes total cellular RNA. In other embodiments, certain abundantRNA sequences (e.g., ribosomal RNAs) have been depleted. In someembodiments, the poly(A)-tailed mRNA fraction in the total RNApreparation has been enriched. In some embodiments, the cDNA is producedby random-primed cDNA synthesis methods. In other embodiments, the cDNAsynthesis is initiated at the poly(A) tail of mature mRNAs by priming byoligo(dT)-containing oligonucleotides. Methods for depletion, poly(A)enrichment, and cDNA synthesis are well known to those skilled in theart.

The method can further include amplifying the nucleic acid sample (e.g.,DNA or RNA sample) by specific or non-specific nucleic acidamplification methods that are well known to those skilled in the art.In some embodiments, certain embodiments, the nucleic acid sample isamplified, e.g., by whole-genome amplification methods such asrandom-primed strand-displacement amplification.

In other embodiments, the nucleic acid sample is fragmented or shearedby physical or enzymatic methods and ligated to synthetic adapters,size-selected (e.g., by preparative gel electrophoresis) and amplified(e.g., by PCR). In other embodiments, the fragmented and adapter-ligatedgroup of nucleic acids is used without explicit size selection oramplification prior to hybrid selection.

In other embodiments, the isolated DNA (e.g., the genomic DNA) isfragmented or sheared. In some embodiments, the library includes lessthan 50% of genomic DNA, such as a subfraction of genomic DNA that is areduced representation or a defined portion of a genome, e.g., that hasbeen subfractionated by other means. In other embodiments, the libraryincludes all or substantially all genomic DNA.

In some embodiments, the library includes less than 50% of genomic DNA,such as a subfraction of genomic DNA that is a reduced representation ora defined portion of a genome, e.g., that has been subfractionated byother means. In other embodiments, the library includes all orsubstantially all genomic DNA. Protocols for isolating and preparinglibraries from whole genomic or subgenomic fragments are known in theart (e.g., Illumina's genomic DNA sample preparation kit). AlternativeDNA shearing methods can be more automatable and/or more efficient(e.g., with degraded FFPE samples). Alternatives to DNA shearing methodscan also be used to avoid a ligation step during library preparation.

The methods described herein can be performed using a small amount ofnucleic acids, e.g., when the amount of source DNA is limiting (e.g.,even after whole-genome amplification). In one embodiment, the nucleicacid comprises less than about 5 μg, 4 μg, 3 μg, 2 μg, 1 μg, 0.8 μg, 0.7μg, 0.6 μg, 0.5 μg, or 400 ng, 300 ng, 200 ng, 100 ng, 50 ng, or 20 ngor less of nucleic acid sample. For example, to prepare 500 ng ofhybridization-ready nucleic acids, one typically begins with 3 μg ofgenomic DNA. One can start with less, however, if one amplifies thegenomic DNA (e.g., using PCR) before the step of solution hybridization.Thus it is possible, but not essential, to amplify the genomic DNAbefore solution hybridization.

In some embodiments, a library is generated using DNA (e.g., genomicDNA) from a sample tissue, and a corresponding library is generated withRNA (or cDNA) isolated from the same sample tissue.

Design of Baits

A bait can be a nucleic acid molecule, e.g., a DNA or RNA molecule,which can hybridize to (e.g., be complementary to), and thereby allowcapture of a target nucleic acid. In one embodiment, a bait is an RNAmolecule. In other embodiments, a bait includes a binding entity, e.g.,an affinity tag, that allows capture and separation, e.g., by binding toa binding entity, of a hybrid formed by a bait and a nucleic acidhybridized to the bait. In one embodiment, a bait is suitable forsolution phase hybridization.

Baits can be produced and used by methods and hybridization conditionsas described in US 2010/0029498 and Gnirke, A. et al. (2009) NatBiotechnol. 27(2):182-189, and U.S. Ser. No. 61/428,568, filed Dec. 30,2010, incorporated herein by reference. For example, biotinylated RNAbaits can be produced by obtaining a pool of synthetic longoligonucleotides, originally synthesized on a microarray, and amplifyingthe oligonucleotides to produce the bait sequences. In some embodiments,the baits are produced by adding an RNA polymerase promoter sequence atone end of the bait sequences, and synthesizing RNA sequences using RNApolymerase. In one embodiment, libraries of syntheticoligodeoxynucleotides can be obtained from commercial suppliers, such asAgilent Technologies, Inc., and amplified using known nucleic acidamplification methods.

Each bait sequence can include a target-specific (e.g., amember-specific) bait sequence and universal tails on each end. As usedherein, the term “bait sequence” can refer to the target-specific baitsequence or the entire oligonucleotide including the target-specific“bait sequence” and other nucleotides of the oligonucleotide.

In one embodiment, the bait is an oligonucleotide about 200 nucleotidesin length, of which 170 nucleotides are target-specific “bait sequence”.The other 30 nucleotides (e.g., 15 nucleotides on each end) areuniversal arbitrary tails used for PCR amplification. The tails can beany sequence selected by the user. The bait sequences described hereincan be used for selection of exons and short target sequences. In oneembodiment, the bait is between about 100 nucleotides and 300nucleotides in length. In another embodiment, the bait is between about130 nucleotides and 230 nucleotides in length. In yet anotherembodiment, the bait is between about 150 nucleotides and 200nucleotides in length. The target-specific sequences in the baits, e.g.,for selection of exons and short target sequences, are between about 40nucleotides and 1000 nucleotides in length. In one embodiment, thetarget-specific sequence is between about 70 nucleotides and 300nucleotides in length. In another embodiment, the target-specificsequence is between about 100 nucleotides and 200 nucleotides in length.In yet another embodiment, the target-specific sequence is between about120 nucleotides and 170 nucleotides in length.

Sequencing

The invention also includes methods of sequencing nucleic acids. In oneembodiment, any of a variety of sequencing reactions known in the artcan be used to directly sequence at least a portion of a fusion. In oneembodiment, the fusion sequence is compared to a corresponding reference(control) sequence.

In one embodiment, the sequence of the fusion nucleic acid molecule isdetermined by a method that includes one or more of: hybridizing anoligonucleotide, e.g., an allele specific oligonucleotide for onealteration described herein to said nucleic acid; hybridizing a primer,or a primer set (e.g., a primer pair), that amplifies a regioncomprising the mutation or a fusion junction of the allele; amplifying,e.g., specifically amplifying, a region comprising the mutation or afusion junction of the allele; attaching an adapter oligonucleotide toone end of a nucleic acid that comprises the mutation or a fusionjunction of the allele; generating an optical, e.g., a colorimetricsignal, specific to the presence of the one of the mutation or fusionjunction; hybridizing a nucleic acid comprising the mutation or fusionjunction to a second nucleic acid, e.g., a second nucleic acid attachedto a substrate; generating a signal, e.g., an electrical or fluorescentsignal, specific to the presence of the mutation or fusion junction; andincorporating a nucleotide into an oligonucleotide that is hybridized toa nucleic acid that contains the mutation or fusion junction.

In another embodiment, the sequence is determined by a method thatcomprises one or more of: determining the nucleotide sequence from anindividual nucleic acid molecule, e.g., where a signal corresponding tothe sequence is derived from a single molecule as opposed, e.g., from asum of signals from a plurality of clonally expanded molecules;determining the nucleotide sequence of clonally expanded proxies forindividual nucleic acid molecules; massively parallel short-readsequencing; template-based sequencing; pyrosequencing; real-timesequencing comprising imaging the continuous incorporation ofdye-labeling nucleotides during DNA synthesis; nanopore sequencing;sequencing by hybridization; nano-transistor array based sequencing;polony sequencing; scanning tunneling microscopy (STM) based sequencing;or nanowire-molecule sensor based sequencing.

Any method of sequencing known in the art can be used. Exemplarysequencing reactions include those based on techniques developed byMaxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger(Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). Any of a variety ofautomated sequencing procedures can be utilized when performing theassays (Biotechniques (1995) 19:448), including sequencing by massspectrometry (see, for example, U.S. Pat. No. 5,547,835 andinternational patent application Publication Number WO 94/16101,entitled DNA Sequencing by Mass Spectrometry by H. Koster; U.S. Pat. No.5,547,835 and international patent application Publication Number WO94/21822 entitled DNA Sequencing by Mass Spectrometry Via ExonucleaseDegradation by H. Koster), and U.S. Pat. No. 5,605,798 and InternationalPatent Application No. PCT/US96/03651 entitled DNA Diagnostics Based onMass Spectrometry by H. Köster; Cohen et al. (1996) Adv Chromatogr36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159).

Sequencing of nucleic acid molecules can also be carried out usingnext-generation sequencing (NGS). Next-generation sequencing includesany sequencing method that determines the nucleotide sequence of eitherindividual nucleic acid molecules or clonally expanded proxies forindividual nucleic acid molecules in a highly parallel fashion (e.g.,greater than 10⁵ molecules are sequenced simultaneously). In oneembodiment, the relative abundance of the nucleic acid species in thelibrary can be estimated by counting the relative number of occurrencesof their cognate sequences in the data generated by the sequencingexperiment. Next generation sequencing methods are known in the art, andare described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews11:31-46, incorporated herein by reference.

In one embodiment, the next-generation sequencing allows for thedetermination of the nucleotide sequence of an individual nucleic acidmolecule (e.g., Helicos BioSciences' HeliScope Gene Sequencing system,and Pacific Biosciences' PacBio RS system). In other embodiments, thesequencing method determines the nucleotide sequence of clonallyexpanded proxies for individual nucleic acid molecules (e.g., the Solexasequencer, Illumina Inc., San Diego, Calif; 454 Life Sciences (Branford,Conn.), and Ion Torrent). e.g., massively parallel short-read sequencing(e.g., the Solexa sequencer, Illumina Inc., San Diego, Calif.), whichgenerates more bases of sequence per sequencing unit than othersequencing methods that generate fewer but longer reads. Other methodsor machines for next-generation sequencing include, but are not limitedto, the sequencers provided by 454 Life Sciences (Branford, Conn.),Applied Biosystems (Foster City, Calif.; SOLiD sequencer), and HelicosBioSciences Corporation (Cambridge, Mass.).

Platforms for next-generation sequencing include, but are not limitedto, Roche/454's Genome Sequencer (GS) FLX System, Illumina/Solexa'sGenome Analyzer (GA), Life/APG's Support Oligonucleotide LigationDetection (SOLiD) system, Polonator's G.007 system, Helicos BioSciences'HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RSsystem.

NGS technologies can include one or more of steps, e.g., templatepreparation, sequencing and imaging, and data analysis.

Template Preparation

Methods for template preparation can include steps such as randomlybreaking nucleic acids (e.g., genomic DNA or cDNA) into smaller sizesand generating sequencing templates (e.g., fragment templates ormate-pair templates). The spatially separated templates can be attachedor immobilized to a solid surface or support, allowing massive amountsof sequencing reactions to be performed simultaneously. Types oftemplates that can be used for NGS reactions include, e.g., clonallyamplified templates originating from single DNA molecules, and singleDNA molecule templates.

Methods for preparing clonally amplified templates include, e.g.,emulsion PCR (emPCR) and solid-phase amplification.

EmPCR can be used to prepare templates for NGS. Typically, a library ofnucleic acid fragments is generated, and adapters containing universalpriming sites are ligated to the ends of the fragment. The fragments arethen denatured into single strands and captured by beads. Each beadcaptures a single nucleic acid molecule. After amplification andenrichment of emPCR beads, a large amount of templates can be attachedor immobilized in a polyacrylamide gel on a standard microscope slide(e.g., Polonator), chemically crosslinked to an amino-coated glasssurface (e.g., Life/APG; Polonator), or deposited into individualPicoTiterPlate (PTP) wells (e.g., Roche/454), in which the NGS reactioncan be performed.

Solid-phase amplification can also be used to produce templates for NGS.Typically, forward and reverse primers are covalently attached to asolid support. The surface density of the amplified fragments is definedby the ratio of the primers to the templates on the support. Solid-phaseamplification can produce hundreds of millions spatially separatedtemplate clusters (e.g., Illumina/Solexa). The ends of the templateclusters can be hybridized to universal sequencing primers for NGSreactions.

Other methods for preparing clonally amplified templates also include,e.g., Multiple Displacement Amplification (MDA) (Lasken R. S. Curr OpinMicrobiol. 2007; 10(5):510-6). MDA is a non-PCR based DNA amplificationtechnique. The reaction involves annealing random hexamer primers to thetemplate and DNA synthesis by high fidelity enzyme, typically Φ29 at aconstant temperature. MDA can generate large sized products with lowererror frequency.

Template amplification methods such as PCR can be coupled with NGSplatforms to target or enrich specific regions of the genome (e.g.,exons). Exemplary template enrichment methods include, e.g.,microdroplet PCR technology (Tewhey R. et al., Nature Biotech. 2009,27:1025-1031), custom-designed oligonucleotide microarrays (e.g.,Roche/NimbleGen oligonucleotide microarrays), and solution-basedhybridization methods (e.g., molecular inversion probes (MIPs) (PorrecaG. J. et al., Nature Methods, 2007, 4:931-936; Krishnakumar S. et al.,Proc. Natl. Acad. Sci. USA, 2008, 105:9296-9310; Turner E. H. et al.,Nature Methods, 2009, 6:315-316), and biotinylated RNA capture sequences(Gnirke A. et al., Nat. Biotechnol. 2009; 27(2):182-17)

Single-molecule templates are another type of templates that can be usedfor NGS reaction. Spatially separated single molecule templates can beimmobilized on solid supports by various methods. In one approach,individual primer molecules are covalently attached to the solidsupport. Adapters are added to the templates and templates are thenhybridized to the immobilized primers. In another approach,single-molecule templates are covalently attached to the solid supportby priming and extending single-stranded, single-molecule templates fromimmobilized primers. Universal primers are then hybridized to thetemplates. In yet another approach, single polymerase molecules areattached to the solid support, to which primed templates are bound.

Sequencing and Imaging

Exemplary sequencing and imaging methods for NGS include, but are notlimited to, cyclic reversible termination (CRT), sequencing by ligation(SBL), single-molecule addition (pyrosequencing), and real-timesequencing.

CRT uses reversible terminators in a cyclic method that minimallyincludes the steps of nucleotide incorporation, fluorescence imaging,and cleavage. Typically, a DNA polymerase incorporates a singlefluorescently modified nucleotide corresponding to the complementarynucleotide of the template base to the primer. DNA synthesis isterminated after the addition of a single nucleotide and theunincorporated nucleotides are washed away. Imaging is performed todetermine the identity of the incorporated labeled nucleotide. Then inthe cleavage step, the terminating/inhibiting group and the fluorescentdye are removed. Exemplary NGS platforms using the CRT method include,but are not limited to, Illumina/Solexa Genome Analyzer (GA), which usesthe clonally amplified template method coupled with the four-color CRTmethod detected by total internal reflection fluorescence (TIRF); andHelicos BioSciences/HeliScope, which uses the single-molecule templatemethod coupled with the one-color CRT method detected by TIRF.

SBL uses DNA ligase and either one-base-encoded probes ortwo-base-encoded probes for sequencing. Typically, a fluorescentlylabeled probe is hybridized to its complementary sequence adjacent tothe primed template. DNA ligase is used to ligate the dye-labeled probeto the primer. Fluorescence imaging is performed to determine theidentity of the ligated probe after non-ligated probes are washed away.The fluorescent dye can be removed by using cleavable probes toregenerate a 5′-PO₄ group for subsequent ligation cycles. Alternatively,a new primer can be hybridized to the template after the old primer isremoved. Exemplary SBL platforms include, but are not limited to,Life/APG/SOLiD (support oligonucleotide ligation detection), which usestwo-base-encoded probes.

Pyrosequencing method is based on detecting the activity of DNApolymerase with another chemiluminescent enzyme. Typically, the methodallows sequencing of a single strand of DNA by synthesizing thecomplementary strand along it, one base pair at a time, and detectingwhich base was actually added at each step. The template DNA isimmobile, and solutions of A, C, G, and T nucleotides are sequentiallyadded and removed from the reaction. Light is produced only when thenucleotide solution complements the first unpaired base of the template.The sequence of solutions which produce chemiluminescent signals allowsthe determination of the sequence of the template. Exemplarypyrosequencing platforms include, but are not limited to, Roche/454,which uses DNA templates prepared by emPCR with 1-2 million beadsdeposited into PTP wells.

Real-time sequencing involves imaging the continuous incorporation ofdye-labeled nucleotides during DNA synthesis. Exemplary real-timesequencing platforms include, but are not limited to, PacificBiosciences platform, which uses DNA polymerase molecules attached tothe surface of individual zero-mode waveguide (ZMW) detectors to obtainsequence information when phospholinked nucleotides are beingincorporated into the growing primer strand; Life/VisiGen platform,which uses an engineered DNA polymerase with an attached fluorescent dyeto generate an enhanced signal after nucleotide incorporation byfluorescence resonance energy transfer (FRET); and LI-COR Biosciencesplatform, which uses dye-quencher nucleotides in the sequencingreaction.

Other sequencing methods for NGS include, but are not limited to,nanopore sequencing, sequencing by hybridization, nano-transistor arraybased sequencing, polony sequencing, scanning tunneling microscopy (STM)based sequencing, and nanowire-molecule sensor based sequencing.

Nanopore sequencing involves electrophoresis of nucleic acid moleculesin solution through a nano-scale pore which provides a highly confinedspace within which single-nucleic acid polymers can be analyzed.Exemplary methods of nanopore sequencing are described, e.g., in BrantonD. et al., Nat Biotechnol. 2008; 26(10):1146-53.

Sequencing by hybridization is a non-enzymatic method that uses a DNAmicroarray. Typically, a single pool of DNA is fluorescently labeled andhybridized to an array containing known sequences. Hybridization signalsfrom a given spot on the array can identify the DNA sequence. Thebinding of one strand of DNA to its complementary strand in the DNAdouble-helix is sensitive to even single-base mismatches when the hybridregion is short or is specialized mismatch detection proteins arepresent. Exemplary methods of sequencing by hybridization are described,e.g., in Hanna G. J. et al., J. Clin. Microbiol. 2000; 38 (7): 2715-21;and Edwards J. R. et al., Mut. Res. 2005; 573 (1-2): 3-12.

Polony sequencing is based on polony amplification andsequencing-by-synthesis via multiple single-base-extensions (FISSEQ).Polony amplification is a method to amplify DNA in situ on apolyacrylamide film. Exemplary polony sequencing methods are described,e.g., in US Patent Application Publication No. 2007/0087362.

Nano-transistor array based devices, such as Carbon NanoTube FieldEffect Transistor (CNTFET), can also be used for NGS. For example, DNAmolecules are stretched and driven over nanotubes by micro-fabricatedelectrodes. DNA molecules sequentially come into contact with the carbonnanotube surface, and the difference in current flow from each base isproduced due to charge transfer between the DNA molecule and thenanotubes. DNA is sequenced by recording these differences. ExemplaryNano-transistor array based sequencing methods are described, e.g., inU.S. Patent Application Publication No. 2006/0246497.

Scanning tunneling microscopy (STM) can also be used for NGS. STM uses apiezo-electric-controlled probe that performs a raster scan of aspecimen to form images of its surface. STM can be used to image thephysical properties of single DNA molecules, e.g., generating coherentelectron tunneling imaging and spectroscopy by integrating scanningtunneling microscope with an actuator-driven flexible gap. Exemplarysequencing methods using STM are described, e.g., in U.S. PatentApplication Publication No. 2007/0194225.

A molecular-analysis device which is comprised of a nanowire-moleculesensor can also be used for NGS. Such device can detect the interactionsof the nitrogenous material disposed on the nanowires and nucleic acidmolecules such as DNA. A molecule guide is configured for guiding amolecule near the molecule sensor, allowing an interaction andsubsequent detection. Exemplary sequencing methods usingnanowire-molecule sensor are described, e.g., in U.S. Patent ApplicationPublication No. 2006/0275779.

Double ended sequencing methods can be used for NGS. Double endedsequencing uses blocked and unblocked primers to sequence both the senseand antisense strands of DNA. Typically, these methods include the stepsof annealing an unblocked primer to a first strand of nucleic acid;annealing a second blocked primer to a second strand of nucleic acid;elongating the nucleic acid along the first strand with a polymerase;terminating the first sequencing primer; deblocking the second primer;and elongating the nucleic acid along the second strand. Exemplarydouble ended sequencing methods are described, e.g., in U.S. Pat. No.7,244,567.

Data Analysis

After NGS reads have been generated, they can be aligned to a knownreference sequence or assembled de novo.

For example, identifying genetic variations such as single-nucleotidepolymorphism and structural variants in a sample (e.g., a tumor sample)can be accomplished by aligning NGS reads to a reference sequence (e.g.,a wild-type sequence). Methods of sequence alignment for NGS aredescribed e.g., in Trapnell C. and Salzberg S. L. Nature Biotech., 2009,27:455-457.

Examples of de novo assemblies are described, e.g., in Warren R. et al.,Bioinformatics, 2007, 23:500-501; Butler J. et al., Genome Res., 2008,18:810-820; and Zerbino D. R. and Birney E., Genome Res., 2008,18:821-829.

Sequence alignment or assembly can be performed using read data from oneor more NGS platforms, e.g., mixing Roche/454 and Illumina/Solexa readdata.

Algorithms and methods for data analysis are described in U.S. Ser. No.61/428,568, filed Dec. 30, 2010, incorporated herein by reference.

Fusion Expression Level

In certain embodiments, fusion expression level can also be assayed.Fusion expression can be assessed by any of a wide variety of methodsfor detecting expression of a transcribed molecule or protein.Non-limiting examples of such methods include immunological methods fordetection of secreted, cell-surface, cytoplasmic, or nuclear proteins,protein purification methods, protein function or activity assays,nucleic acid hybridization methods, nucleic acid reverse transcriptionmethods, and nucleic acid amplification methods.

In certain embodiments, activity of a particular gene is characterizedby a measure of gene transcript (e.g., mRNA), by a measure of thequantity of translated protein, or by a measure of gene productactivity. fusion expression can be monitored in a variety of ways,including by detecting mRNA levels, protein levels, or protein activity,any of which can be measured using standard techniques. Detection caninvolve quantification of the level of gene expression (e.g., genomicDNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can bea qualitative assessment of the level of gene expression, in particularin comparison with a control level. The type of level being detectedwill be clear from the context.

Methods of detecting and/or quantifying the fusion gene transcript (mRNAor cDNA made therefrom) using nucleic acid hybridization techniques areknown to those of skill in the art (see Sambrook et al. supra). Forexample, one method for evaluating the presence, absence, or quantity ofcDNA involves a Southern transfer as described above. Briefly, the mRNAis isolated (e.g., using an acid guanidinium-phenol-chloroformextraction method, Sambrook et al. supra.) and reverse transcribed toproduce cDNA. The cDNA is then optionally digested and run on a gel inbuffer and transferred to membranes. Hybridization is then carried outusing the nucleic acid probes specific for the fusion cDNA, e.g., usingthe probes and primers described herein.

In other embodiments, expression is assessed by preparing genomic DNA ormRNA/cDNA (i.e., a transcribed polynucleotide) from cells in a subjectsample, and by hybridizing the genomic DNA or mRNA/cDNA with a referencepolynucleotide which is a complement of a polynucleotide comprising thefusion, and fragments thereof. cDNA can, optionally, be amplified usingany of a variety of polymerase chain reaction methods prior tohybridization with the reference polynucleotide. Expression of a fusionas described herein can likewise be detected using quantitative PCR(QPCR) to assess the level of expression.

Detection of Fusion Polypeptide

The activity or level of a fusion polypeptide can also be detectedand/or quantified by detecting or quantifying the expressed polypeptide.The fusion polypeptide can be detected and quantified by any of a numberof means known to those of skill in the art. These can include analyticbiochemical methods such as electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, or variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, immunohistochemistry (IHC)and the like. A skilled artisan can adapt known protein/antibodydetection methods.

Another agent for detecting a fusion polypeptide is an antibody moleculecapable of binding to a polypeptide corresponding to a marker, e.g., anantibody with a detectable label. Techniques for generating antibodiesare described herein. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin.

In another embodiment, the antibody is labeled, e.g., a radio-labeled,chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody. Inanother embodiment, an antibody derivative (e.g., an antibody conjugatedwith a substrate or with the protein or ligand of a protein-ligand pair{e.g., biotin-streptavidin}), or an antibody fragment (e.g., asingle-chain antibody, an isolated antibody hypervariable domain, etc.)which binds specifically with a fusion protein, is used.

Fusion polypeptides from cells can be isolated using techniques that areknown to those of skill in the art. The protein isolation methodsemployed can, for example, be such as those described in Harlow and Lane(Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York).

Means of detecting proteins using electrophoretic techniques are wellknown to those of skill in the art (see generally, R. Scopes (1982)Protein Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methodsin Enzymology Vol. 182: Guide to Protein Purification, Academic Press,Inc., N.Y.).

In another embodiment, Western blot (immunoblot) analysis is used todetect and quantify the presence of a polypeptide in the sample.

In another embodiment, the polypeptide is detected using an immunoassay.As used herein, an immunoassay is an assay that utilizes an antibody tospecifically bind to the analyte. The immunoassay is thus characterizedby detection of specific binding of a polypeptide to an anti-antibody asopposed to the use of other physical or chemical properties to isolate,target, and quantify the analyte.

The fusion polypeptide is detected and/or quantified using any of anumber of immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Asai (1993) Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;Stites & Ten (1991) Basic and Clinical Immunology 7th Edition.

Kits

In one aspect, the invention features, a kit, e.g., containing anoligonucleotide having a mutation described herein, e.g., a fusion.Optionally, the kit can also contain an oligonucleotide that is thewildtype counterpart of the mutant oligonucleotide.

A kit featured in the invention can include a carrier, e.g., a meansbeing compartmentalized to receive in close confinement one or morecontainer means. In one embodiment the container contains anoligonucleotide, e.g., a primer or probe as described above. Thecomponents of the kit are useful, for example, to diagnose or identify amutation in a tumor sample in a patient. The probe or primer of the kitcan be used in any sequencing or nucleotide detection assay known in theart, e.g., a sequencing assay, e.g., an NGS method, RT-PCR, or in situhybridization.

In some embodiments, the components of the kit are useful, for example,to diagnose or identify a fusion in a tumor sample in a patient, and toaccordingly identify an appropriate therapeutic agent to treat thecancer.

A kit featured in the invention can include, e.g., assay positive andnegative controls, nucleotides, enzymes (e.g., RNA or DNA polymerase orligase), solvents or buffers, a stabilizer, a preservative, a secondaryantibody, e.g., an anti-HRP antibody (IgG) and a detection reagent.

An oligonucleotide can be provided in any form, e.g., liquid, dried,semi-dried, or lyophilized, or in a form for storage in a frozencondition.

Typically, an oligonucleotide, and other components in a kit areprovided in a form that is sterile. An oligonucleotide, e.g., anoligonucleotide that contains an mutation, e.g., a fusion, describedherein, or an oligonucleotide complementary to a fusion describedherein, is provided in a liquid solution, the liquid solution generallyis an aqueous solution, e.g., a sterile aqueous solution. When theoligonucleotide is provided as a dried form, reconstitution generally isaccomplished by the addition of a suitable solvent. The solvent, e.g.,sterile buffer, can optionally be provided in the kit.

The kit can include one or more containers for the compositioncontaining an oligonucleotide in a concentration suitable for use in theassay or with instructions for dilution for use in the assay. In someembodiments, the kit contains separate containers, dividers orcompartments for the oligonucleotide and assay components, and theinformational material. For example, the oligonucleotides can becontained in a bottle or vial, and the informational material can becontained in a plastic sleeve or packet. In other embodiments, theseparate elements of the kit are contained within a single, undividedcontainer. For example, an oligonucleotide composition is contained in abottle or vial that has attached thereto the informational material inthe form of a label. In some embodiments, the kit includes a plurality(e.g., a pack) of individual containers, each containing one or moreunit forms (e.g., for use with one assay) of an oligonucleotide. Forexample, the kit includes a plurality of ampoules, foil packets, orblister packs, each containing a single unit of oligonucleotide for usein sequencing or detecting a mutation in a tumor sample. The containersof the kits can be air tight and/or waterproof. The container can belabeled for use.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds to a fusion polypeptide;and, optionally, (2) a second, different antibody which binds to eitherthe polypeptide or the first antibody and is conjugated to a detectableagent.

In one embodiment, the kit can include informational material forperforming and interpreting the sequencing or diagnostic. In anotherembodiment, the kit can provide guidance as to where to report theresults of the assay, e.g., to a treatment center or healthcareprovider. The kit can include forms for reporting the results of asequencing or diagnostic assay described herein, and address and contactinformation regarding where to send such forms or other relatedinformation; or a URL (Uniform Resource Locator) address for reportingthe results in an online database or an online application (e.g., anapp). In another embodiment, the informational material can includeguidance regarding whether a patient should receive treatment with aparticular chemotherapeutic drug, depending on the results of the assay.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawings, and/or photographs,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about the sequencingor diagnostic assay and/or its use in the methods described herein. Theinformational material can also be provided in any combination offormats.

In some embodiments, a biological sample is provided to an assayprovider, e.g., a service provider (such as a third party facility) or ahealthcare provider, who evaluates the sample in an assay and provides aread out. For example, in one embodiment, an assay provider receives abiological sample from a subject, such as a blood or tissue sample,e.g., a biopsy sample, and evaluates the sample using an assay describedherein, e.g., a sequencing assay or in situ hybridization assay, anddetermines that the sample contains a fusion. The assay provider, e.g.,a service provider or healthcare provider, can then conclude that thesubject is, or is not, a candidate for a particular drug or a particularcancer treatment regimen.

Exemplary Rearrangements

TABLE 1 BICC1- This is an in-frame fusion (chr10 inversion). The FGFR2breakpoint in FGFR2 is found approximately in the middle of the kinasedomain. This was selected since the breakpoint is close to a rare exonthat was baited. There are possibly additional breakpoints. FGFR2- Thisis a chr10 deletion. The breakpoint is KIAA1598 in the 3′ utr of FGFR2so the entire protein is intact. This is similar to the FGFR3-TACC3structure. FGFR2- This is an in-frame fusion (chr4; 10 translocation).The TACC3 breakpoints are in FGFR2 intron 17 and TACC3 intron10. TheFGFR2 brkpt is right after the kinase domain. FGFR3-TACC3 has beenrecently reported as a potential driver in GBM (e.g., PMID: 22837387).RABGAP1L- This is an in-frame fusion (chr1 tandem duplication). NTRK1Again the breakpoint found within the tyrosine kinase domain. Thisrearrangement is complex. NTRK1 is also amplified.

Additional description of the alterations disclosed herein in providedin FIGS. 1A-1C and FIGS. 2-17 , which are summarized below.

Fusion Description FGFR2-TACC3 chr10: chr4 translocation FGFR2-KIAA1598chr10 deletion BICC1-FGFR2 chr10 inversion FGFR2-BICC1 chr10: inversion

Genomic Location

Fusion Breakpoint 1 Breakpoint 2 FGFR2- FGFR2(NM_001144915):TACC3(NM_006342): TACC3 chr10: 123,243,122; intron16 chr4: 1,740,657;intron10 FGFR2- FGFR2(NM_001144915): KIAA1598(NM_001127211): KIAA1598chr10: 123,239,241; chr10: 118708643; intron 16 intron6 BICC1-BICC1(NM_001080512): FGFR2(NM_001144915): FGFR2 chr10: 60446461; chr10:123,241,845; intron2 intron 16 FGFR2- FGFR2(NM_001144915):BICC1(NM_001080512): BICC1 chr10: 123,241,713; intron16 chr10:60,567,607; intron 17

Exons

Fusion Exons FGFR2-TACC3 FGFR2 (exon 1-16) − TACC3 (exon11-16)FGFR2-KIAA1598 FGFR2 (exon 1-16) − KIAA1598 (exon7-17) BICC1-FGFR2 BICC1(exon 1-2) − FGFR2 (exon17) FGFR2-BICC1 FGFR2 (exon 1-16) −BICC1(exon18-21)

Exons in the 5′-partner and the 3′-partner

Fusion 5′ Partner 3′ Partner FGFR2-TACC3 FGFR2: kinase domain TACC3:coiled-coil exon 10-16, included region exon 11-16, included in fusionproduct in fusion product FGFR2- FGFR2: kinase KIAA1598 KIAA1598 domainexon 10-16, included in fusion product BICC1-FGFR2 BICC1: unknownfunction FGFR2: kinase domain in fusion product exon 10-16, not includedin fusion product FGFR2-BICC1 FGFR2: kinase domain BICC1: included exon10-16, in fusion product

The RefSeq Gene are databased at UCSC Genome Browser(http://genome.ucsc.edu/cgi-bin/hgc?hgsid=309144129&c=chr4&o=1795038&t=1810599&g=refGene&i=NM_000142

Fusion 5′ Partner 3′ Partner FGFR2-TACC3 FGFR2: NM_001144915 TACC3:NM_006342 FGFR2-KIAA1598 FGFR2: NM_001144915 KIAA1598: NM_001127211BICC1-FGFR2 BICC1: NM_001080512 FGFR2: NM_001144915 FGFR2-BICC1 FGFR2:NM_001144915 BICC1: NM_001080512

The invention is further illustrated by the following example, whichshould not be construed as further limiting.

EXAMPLE

Sequencing of approximately 30 cholangiocarcinomas has revealed 3 FGFR2fusions, 1 FGFR2 amplification and 1 NTRAK 1 fusion. Hepatocellularcarcinomas are far more common worldwide. HCC is a tumor derived fromhepatocytes and ICC is derived from the intrahepatic bile ductepithelium (also known as the cholangiole. Both HCC and ICC are relatedto hepatitis C infection. As these rearrangements were not selectedthrough hybridization capture reaction it is believed thatrearrangements of this type are far more common in these cancers thanthe observed frequency.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims.

1-47. (canceled)
 48. A method of determining presence of a fibroblastgrowth factor receptor (FGFR) gene fusion or a neurotrophic tyrosinereceptor kinase (NTRK) gene fusion, comprising: detecting an FGFR orNTRK gene fusion in an FGFR or NTRK nucleic acid molecule or polypeptidein a sample obtained from a subject that has or is at risk of having acholangiocarcinoma, thereby determining that the FGFR or NTRK genefusion is present in the sample; and responsive to the determination ofthe presence of the FGFR or NTRK gene fusion in the sample, generating areport comprising one or more therapeutic options comprising atherapeutic agent that antagonizes or inhibits an FGFR gene or geneproduct or an NTRK gene or gene product.
 49. The method of claim 48,further comprising administering to the subject an effective amount ofthe therapeutic agent.
 50. The method of claim 48, wherein saidtherapeutic agent comprises one or more of: a kinase inhibitor; atyrosine kinase inhibitor; a pan-FGFR2 inhibitor; a pan-TRK inhibitor; amulti-specific kinase inhibitor; an FGF receptor inhibitor; a reversibleor an irreversible FGFR2 inhibitor; a small molecule that binds to theFGFR2 gene product or the NTRK1 gene product; an antibody moleculeagainst the FGFR2 gene product or the NTRK1 gene product; a kinaseinhibitor that is selective for the FGFR2 gene product or the NTRK1 geneproduct; and a siRNA, antisense RNA, or other nucleic acid basedinhibitor of the FGFR2 gene or gene product or the NTRK1 gene or geneproduct.
 51. The method of claim 48, wherein said therapeutic agentcomprises one or more of AZD-2171, AZD-4547, BGJ398, BIBF1120, Brivanib,Cediranib, Dovitinib, ENMD-2076, JNJ 42756493, Masitinib, Lenvatinib,LY2874455, Ponatinib, Pazopanib, R406, Regorafenib, PD173074, PD-173955,Danusertib, Dovitinib Dilactic Acid, TSU-68, Tyrphostin AG 1296,MK-2461, Brivanib Alaninate, Lestaurtinib, PHA-848125, K252a, AZ-23,Oxindole-3, AV369b, ACTB1003, Volasertib, R1530, Loxo-101, ARRY-470,ARRY-786, RXDX-101, RXDX-102, axitinib, bosutinib, dasatinib, erlotinib,gefitinib, imatinib, lapatinib, neratinib, nilotinib, semaxanib,sunitinib, toceranib, vandetanib, vatalanib, sorafenib, PCI-32765,AC220, dovitinib lactate, BIBW 2992, SGX523, PF-04217903, PF-02341066,PF-299804, BMS-777607, ABT-869, MP470, AP24534, JNJ-26483327, MGCD265,DCC-2036, BMS-690154, CEP-11981, tivozanib, OSI-930, MM-121, XL-184,XL-647, and XL228.
 52. The method of claim 48, wherein thecholangiocarcinoma is an intrahepatic cholangiocarcinoma (ICC).
 53. Themethod of claim 48, wherein the cholangiocarcinoma is an extrahepaticcholangiocarcinoma.
 54. The method of claim 48, wherein thecholangiocarcinoma is a metastatic cholangiocarcinoma.
 55. The method ofclaim 48, further comprising acquiring the sample from the subject. 56.The method of claim 48, wherein the sample comprises genomic DNA, cDNA,or RNA.
 57. The method of claim 48, wherein the sample comprisescancerous tissue, whole blood, serum, plasma, buccal scrape, sputum,saliva, cerebrospinal fluid, urine, stool, circulating tumor cells,circulating nucleic acids, or bone marrow.
 58. The method of claim 57,wherein the sample is a tissue biopsy sample.
 59. The method of claim57, wherein the sample comprises circulating tumor cells or circulatingnucleic acids.
 60. The method of claim 48, further comprising, prior toacquiring the sequence, enriching the sample for an FGFR or NTRK nucleicacid molecule.
 61. The method of claim 48, wherein the FGFR gene fusionis an FGFR1, FGFR2, or FGFR3 gene fusion.
 62. The method of claim 48,wherein the NTRK gene fusion is an NTRK1 gene fusion.
 63. The method ofclaim 48, wherein detecting the FGFR or NTRK gene fusion comprisessequencing at least nucleic acid molecule that comprises the FGFR orNTRK gene fusion.
 64. The method of claim 63, wherein the sequencing isnext-generation sequencing (NGS).
 65. The method of claim 48, whereinthe sequencing comprises: (a) hybridizing an oligonucleotide specificfor the FGFR or NTRK gene fusion to the FGFR or NTRK nucleic acidmolecule; (b) hybridizing a primer that amplifies a region comprisingthe fusion junction of the FGFR or NTRK gene fusion to the FGFR or NTRKnucleic acid molecule; (c) amplifying the region comprising the fusionjunction of the FGFR or NTRK gene fusion; (d) attaching an adapteroligonucleotide to one end of a nucleic acid that comprises the fusionjunction of the FGFR or NTRK gene fusion; (e) generating a signalspecific to the presence of the fusion junction of the FGFR or NTRK genefusion; and/or (f) incorporating a nucleotide into an oligonucleotidethat is hybridized to a nucleic acid that contains the fusion junctionof the FGFR or NTRK gene fusion.
 66. The method of claim 48, wherein theFGFR or NTRK gene fusion is: (a) an FGFR2-TACC3 gene fusion comprisingexons 1-16 of SEQ ID NO: 1 and exons 11-16 of SEQ ID NO: 3; (b) anFGFR2-KIAA1598 gene fusion comprising exons 1-16 of SEQ ID NO: 1 andexons 7-17 of SEQ ID NO: 5; (c) a BICC1-FGFR2 gene fusion comprisingexons 1-2 of SEQ ID NO: 7 and exon 17 of SEQ ID NO: 1; (d) anFGFR2-BICC1 gene fusion comprising exons 1-16 of SEQ ID NO: 1 and exons18-21 of SEQ ID NO: 7; (e) a PARK2-FGFR2 gene fusion comprising exons1-9 of SEQ ID NO: 9 and exon 18 of SEQ ID NO: 11; (f) an FGFR2-NOL4 genefusion comprising exons 1-17 of SEQ ID NO: 11 and exons 7-11 of SEQ IDNO: 13; (g) a ZDHHC6-FGFR2 gene fusion comprising exons 1-5 of SEQ IDNO: 15 and exon 18 of SEQ ID NO: 11; or (h) a RABGAP1L-NTRK1 genefusion.
 67. The method of claim 48, wherein the report is in electronic,web-based, or paper form.
 68. The method of claim 48, further comprisingproviding the report to the patient or to a caregiver, physician,oncologist, hospital, clinic, third-party payor, insurance company, orgovernment office.
 69. The method of claim 48, wherein the reportfurther comprises information on likely effectiveness of a therapeuticoption, acceptability of a therapeutic option, advisability of applyingthe therapeutic option to a patient, information or a recommendation onadministration of the therapeutic agent at a preselected dosage or in apreselected treatment regimen, information or a recommendation onadministration of the therapeutic agent in combination with anotherdrug, or information on the role of an FGFR or NTRK gene fusion or awild-type FGFR or NTRK gene sequence in disease.
 70. A method ofstratifying a subject that has or is at risk of having acholangiocarcinoma for treatment, comprising: detecting presence orabsence of a fibroblast growth factor receptor (FGFR) gene fusion or aneurotrophic tyrosine receptor kinase (NTRK) gene fusion in an FGFR orNTRK nucleic acid molecule or polypeptide in a sample obtained from thesubject; responsive to a determination of the presence of the FGFR orNTRK gene fusion in the sample, classifying the subject as a candidateto receive a treatment comprising a therapeutic agent that antagonizesor inhibits an FGFR gene or gene product or an NTRK gene or geneproduct; and responsive to a determination of the absence of the FGFR orNTRK gene fusion in the sample, classifying the subject as a candidateto receive a treatment other than a therapeutic agent that antagonizesor inhibits an FGFR gene or gene product or an NTRK gene or geneproduct.
 71. A method of identifying a subject that has or is at risk ofhaving a cholangiocarcinoma as likely or unlikely to respond to atreatment, comprising: detecting presence or absence of a fibroblastgrowth factor receptor (FGFR) gene fusion or a neurotrophic tyrosinereceptor kinase (NTRK) gene fusion in an FGFR or NTRK nucleic acidmolecule or polypeptide in a sample obtained from the subject;responsive to a determination of the presence of the FGFR or NTRK genefusion in the sample, identifying the subject as likely to respond to atreatment comprising a therapeutic agent that antagonizes or inhibits anFGFR gene or gene product or an NTRK gene or gene product; andresponsive to a determination of the absence of the FGFR or NTRK genefusion in the sample, identifying the subject as unlikely to respond toa treatment comprising a therapeutic agent that antagonizes or inhibitsan FGFR gene or gene product or an NTRK gene or gene product.